[ccp4bb] AW: [EXTERNAL] [ccp4bb] R: [ccp4bb] Heavy atom vs light atoms density

[Schreuder, Herman /DE]

Dear Vito,

I have also worked with iodinated and brominated compounds and in general the 
light atoms are not flattened out. As John Helliwell pointed out, you may look 
at series termination effects. However, since your map does not appear to be of 
extremely high resolution, I guess all useful data will have been collected and 
included.

The other thing to look at is occupancy. If your ligand is bound at partial 
occupancy, the iodides will still have very strong density, but the lighter 
atoms may fall below the 1 sigma threshold. You can test this by refining a 
group occupancy for your ligand and also by scrolling down the contour level in 
coot to see if some density appears at lower contour levels. 

Good luck!
Herman 

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Vito Calderone
Gesendet: Mittwoch, 10. Juni 2020 10:34
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] R: [ccp4bb] Heavy atom vs light atoms density

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear Pierre,
 I was in particular talking of 2FoFc refinement maps.
In the attached picture for example you can see the 2FoFc map contoured at 1 
sigma level of an I3C molecule bound to a protein where for I3C the only 
visible density is that of iodines whereas the density of the other atoms of 
the ligand is insignificant.
Best regards

Vito

-Messaggio originale-
Da: CCP4 bulletin board  Per conto di LEGRAND Pierre
Inviato: mercoledì 10 giugno 2020 09:31
A: CCP4BB@JISCMAIL.AC.UK
Oggetto: Re: [ccp4bb] Heavy atom vs light atoms density

Dear Vito,
Could you precise in what kind of maps are you experiencing these effects:
2FoFc refinement maps or experimental phasing ?
I had this kind of effects long ago in experiemental phasing due to Fourier 
transform ripple effects. This could be due to scaling problems, low resolution 
truncation or incompletness (maybe intensity overloads).
Another source condition could be local X-ray dose degradation due to the high 
absoption of the metals.
Best regards,
Pierre Legrand
PROXIMA-1, SOLEIL

De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Vito Calderone 
[calder...@cerm.unifi.it] Envoyé : mercredi 10 juin 2020 08:42 À :
CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Heavy atom vs light atoms density

Dear All,
   many of us have probably experienced that, in the diffraction of 
protein ligands containing heavy atoms (cisPt, I3C, etc), the overwhelming 
electron density of the metal can totally flatten that of the light atoms 
around (or rather make it look insignificant).
Is anyone aware of an article/review (to use as a reference) in which this is 
clearly stated/pointed out?
Best regards

Vito Calderone





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[ccp4bb] AW: Molecular replacement problem

[Schreuder, Herman /DE]

Dear Robert,

In addition to the remarks and suggestions by others: How do your electron 
density maps look like? Do they look remotely reasonable, or do they look like 
they had gone through a meat grinder? If they look remotely ok, you could also 
try manual rebuilding, pruning etc. However, if they look chopped, there is no 
sense in trying to (re)build anything in it, either manually or automatically. 
I guess you tried all permutations and variations of P2221, with zero, one and 
two screw axes in all positions?

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Robert S 
Phillips
Gesendet: Donnerstag, 18. Juni 2020 15:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Molecular replacement problem


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] dewar horror stories

[Schreuder, Herman /DE]

I had a similar one-sided discussion with FEDEX about their ignoring of our 
customs declarations for Switzerland. That was then the last Dewar we sent by 
FEDEX.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Jan Dohnalek
Gesendet: Donnerstag, 25. Juni 2020 08:34
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] dewar horror stories


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

We have the suspicion (after several heavy FEDEX failures) they just toss them 
around ... then the neck easily breaks off.
That only explains everything we have seen with completely damaged samples, 
lost, flying around the dewar etc ...
When trying to communicate seriously with FEDEX about these issues - they even 
did not reply ...

Jan



On Wed, Jun 24, 2020 at 9:27 PM Patrick Loll 
mailto:pjl...@gmail.com>> wrote:
Hello community,

We recently had a dry shipping dewar fail catastrophically (while en route to 
the beam line, so, major trauma). I sent it to a company that specializes in 
repair and refurbishing of cryogenic tanks, and they told me it has an internal 
leak, and hence is not reparable. I was expecting that the valve had failed, so 
the internal leak diagnosis came as a surprise.

Has anyone else had a similar experience? Any ideas about how an internal leak 
might come about? The dewar is (was) a Taylor/Wharton CX100, and it was 
traveling in its bespoke shipping case.

Thanks for any insights that might satisfy my curiosity and/or prevent future 
mishaps of this sort.

Cheers,

Pat

__

Patrick J.  Loll, PhD
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(215) 762-7706
pj...@drexel.edu




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--
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic
Tel. +420 325 873 758



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Dear BB,

Since there does not seem a generally accepted term for the subject of this 
discussions, and since even the IUCR scriptures do not give any guidance, I 
would propose to introduce a completely new term:

Measurements per reflection or MPR

This term is politically neutral, should adequately describe this particular 
statistic and is not associated with entrenched traditions at either side of 
the Atlantic.

What do you think?
Herman


Von: CCP4 bulletin board  Im Auftrag von John R Helliwell
Gesendet: Dienstag, 30. Juni 2020 14:34
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear Colleagues,
In an effort to break this naming deadlock, and with Massimo and Ian not 
showing up as yet, I checked the IUCr Dictionary.
“Redundancy“ and “Multiplicity“ are not listed.
The more generic term “Statistical Descriptors“ is though and even offers 
Recommendations:-
http://ww1.iucr.org/iucr-top/comm/cnom/statdes/recomm.html
Point 1, first sentence, fits the various wishes of this thread succinctly, if 
not in a single word, and even not readily allowing an easy acronym.
Greetings,
John

Emeritus Professor John R Helliwell DSc




On 30 Jun 2020, at 13:11, Phil Jeffrey 
mailto:pjeff...@princeton.edu>> wrote:
The people that already use multiplicity are going to find reasons why it's 
the superior naming scheme - although the underlying reason has a lot to do 
with negative associations with 'redundant', perhaps hightened in the current 
environment.  And conversely redundant works for many others - Graeme's 
pragmatic defense of multiplicity actually works both ways - any person who 
takes the trouble to read the stats table, now exiled to Supplementary Data, 
knows what it means.  Surely, then, the only way forward on this almost totally 
irrelevant discussion is to come up with a universally-loathed nomenclature 
that pleases nobody, preferably an acronym whose origins will be lost to 
history and the dusty CCP4 archives (which contain threads similar to this 
one).  I humbly submit:

NFDOF: Nearly Futile Data Overcollection Factor ?
[*]

Or, even better, could we not move on to equally pointless discussions of the 
inappropriateness of "R-factor" ?  I have a long history of rearguard action 
trying to give stupid acronyms a wider audience, so you're guaranteed to hear 
from me on this for years.

(Personally I'm pining for Gerard Kleywegt to resume his quest for overextended 
naming rationales, of which ValLigURL is a personal 'favo[u]rite'.  But I'm 
just old-fashioned.)

Ironically,
Phil Jeffrey
Princeton

[* I too have collected 540 degrees in P1 to solve a SAD structure, just 
because I could, hence "nearly"]
[** The actual answer to this thread is: history is written by the authors of 
scaling programs - and I think the Americans are currently losing at this game, 
thus perilously close to making themselves redundant.]

On 6/30/20 4:14 AM, Winter, Graeme (DLSLtd,RAL,LSCI) wrote:

Or, we could accept the fact that crystallographers are kinda used to 
multiplicity of an individual Miller index being different to multiplicity of 
observations, and in Table 1 know which one you mean? 😉 Given that they add new 
information (at the very least to the scaling model) they are strictly not 
“redundant”.
The amount that anyone outside of methods development cares about the “epsilon” 
multiplicity of reflections is … negligible?
Sorry for chucking pragmatism into a dogmatic debate 😀
Cheerio Graeme



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[ccp4bb] AW: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Dear Bernard and other bulletin board members,

As Gerard mentioned, current data processing programs and table 1’s do not make 
this distinction, but of course, you are free to ask the community to introduce 
it.

My proposal to use “measurements per reflections” is not a joke. It exactly 
describes what is meant by the parameter and it is easily understood even by 
lay people like journal editors and referees, without the need of lengthy 
explanations like the ones we have seen in this thread.

I really would like to ask you to consider replacing 
multiplicity/redundancy/abundancy by MPR. At minimum, it may prevent a thread 
about completeness of data sets to be hijacked by a discussion on whether use 
the name multiplicity of redundancy for the number of measurements per 
reflection.

My 2 cents,
Herman



Von: CCP4 bulletin board  Im Auftrag von Bernhard Rupp
Gesendet: Dienstag, 30. Juni 2020 17:50
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full 
dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

.…but there is a difference whether I measure the same identical hkl over again 
or ‘preferably in more than one symmetry-equivalent position’, to quote the
IUCr. So do we have a MPSR for the same reflection and a MPRR for the related 
reflections?

Cacophonically yours,

BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of John R Helliwell
Sent: Tuesday, June 30, 2020 08:36
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full 
dataset?

Dear Herman,
I think that MPR is a very neat and tidy, excellent, proposal.
Moreover it uses the word “measurements”, and we are an experimental based 
science.
I support it.
Great.
Greetings,
John
Emeritus Professor John R Helliwell DSc




On 30 Jun 2020, at 15:10, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear BB,

Since there does not seem a generally accepted term for the subject of this 
discussions, and since even the IUCR scriptures do not give any guidance, I 
would propose to introduce a completely new term:

Measurements per reflection or MPR

This term is politically neutral, should adequately describe this particular 
statistic and is not associated with entrenched traditions at either side of 
the Atlantic.

What do you think?
Herman


Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von John R Helliwell
Gesendet: Dienstag, 30. Juni 2020 14:34
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

Dear Colleagues,
In an effort to break this naming deadlock, and with Massimo and Ian not 
showing up as yet, I checked the IUCr Dictionary.
“Redundancy“ and “Multiplicity“ are not listed.
The more generic term “Statistical Descriptors“ is though and even offers 
Recommendations:-
http://ww1.iucr.org/iucr-top/comm/cnom/statdes/recomm.html<https://urldefense.proofpoint.com/v2/url?u=http-3A__ww1.iucr.org_iucr-2Dtop_comm_cnom_statdes_recomm.html&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=vb2CFOGKla49hE2sbHAt6LCUz63K7uis9PmSUxUgMcM&s=-45HByHsLJPmc2KRmPKamiFNf1WFCI51GonllFyIRTE&e=>
Point 1, first sentence, fits the various wishes of this thread succinctly, if 
not in a single word, and even not readily allowing an easy acronym.
Greetings,
John

Emeritus Professor John R Helliwell DSc



On 30 Jun 2020, at 13:11, Phil Jeffrey 
mailto:pjeff...@princeton.edu>> wrote:
The people that already use multiplicity are going to find reasons why it's 
the superior naming scheme - although the underlying reason has a lot to do 
with negative associations with 'redundant', perhaps hightened in the current 
environment.  And conversely redundant works for many others - Graeme's 
pragmatic defense of multiplicity actually works both ways - any person who 
takes the trouble to read the stats table, now exiled to Supplementary Data, 
knows what it means.  Surely, then, the only way forward on this almost totally 
irrelevant discussion is to come up with a universally-loathed nomenclature 
that pleases nobody, preferably an acronym whose origins will be lost to 
history and the dusty CCP4 archives (which contain threads similar to this 
one).  I humbly submit:

NFDOF: Nearly Futile Data Overcollection Factor ?
[*]

Or, even better, could we not move on to equally pointless discussions of the 
inappropriateness of "R-factor" ?  I have a long history of rearguard action 
trying to give stupid acronyms a wider audience, so you're guaranteed to hear 
from me on this for years.

(Personally I'm pining for 

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Dear Frank,

in general it is not possible to determine the intensity of a reflection from a 
single fine slice. One needs slices for the complete reflection.
Also, like Bernard, you are imposing criteria on the MPR, which are not imposed 
on the multiplicity/redundancy/abundancy.

All I ask the bulletin to think about my proposal as it is, without prejudices.

Best,
Herman



Von: Frank Von Delft 
Gesendet: Mittwoch, 1. Juli 2020 09:46
An: Schreuder, Herman /DE 
Betreff: Re: [ccp4bb] AW: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to 
get a full dataset?


EXTERNAL : Real sender is 
frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>

If I fine slice the data, does each frame measuring the same reflection (or a 
part of it) count as a measurement?

So that doesn't get us out of the woods, alas.

Sent from tiny silly touch screen
____
From: "Schreuder, Herman /DE" 
mailto:herman.schreu...@sanofi.com>>
Sent: Wednesday, 1 July 2020 08:33
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] AW: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get 
a full dataset?

Dear Bernard and other bulletin board members,

As Gerard mentioned, current data processing programs and table 1’s do not make 
this distinction, but of course, you are free to ask the community to introduce 
it.

My proposal to use “measurements per reflections” is not a joke. It exactly 
describes what is meant by the parameter and it is easily understood even by 
lay people like journal editors and referees, without the need of lengthy 
explanations like the ones we have seen in this thread.

I really would like to ask you to consider replacing 
multiplicity/redundancy/abundancy by MPR. At minimum, it may prevent a thread 
about completeness of data sets to be hijacked by a discussion on whether use 
the name multiplicity of redundancy for the number of measurements per 
reflection.

My 2 cents,
Herman



Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Bernhard Rupp
Gesendet: Dienstag, 30. Juni 2020 17:50
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full 
dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

.…but there is a difference whether I measure the same identical hkl over again 
or ‘preferably in more than one symmetry-equivalent position’, to quote the
IUCr. So do we have a MPSR for the same reflection and a MPRR for the related 
reflections?

Cacophonically yours,

BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of John R Helliwell
Sent: Tuesday, June 30, 2020 08:36
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full 
dataset?

Dear Herman,
I think that MPR is a very neat and tidy, excellent, proposal.
Moreover it uses the word “measurements”, and we are an experimental based 
science.
I support it.
Great.
Greetings,
John
Emeritus Professor John R Helliwell DSc




On 30 Jun 2020, at 15:10, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear BB,

Since there does not seem a generally accepted term for the subject of this 
discussions, and since even the IUCR scriptures do not give any guidance, I 
would propose to introduce a completely new term:

Measurements per reflection or MPR

This term is politically neutral, should adequately describe this particular 
statistic and is not associated with entrenched traditions at either side of 
the Atlantic.

What do you think?
Herman


Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von John R Helliwell
Gesendet: Dienstag, 30. Juni 2020 14:34
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

Dear Colleagues,
In an effort to break this naming deadlock, and with Massimo and Ian not 
showing up as yet, I checked the IUCr Dictionary.
“Redundancy“ and “Multiplicity“ are not listed.
The more generic term “Statistical Descriptors“ is though and even offers 
Recommendations:-
http://ww1.iucr.org/iucr-top/comm/cnom/statdes/recomm.html<https://urldefense.proofpoint.com/v2/url?u=http-3A__ww1.iucr.org_iucr-2Dtop_comm_cnom_statdes_recomm.html&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=vb2CFOGKla49hE2sbHAt6LCUz63K7uis9PmSUxUgMcM&s=-45HByHsLJPmc2KRmPKamiFNf1WFCI51GonllFyIRTE&e=>
Point 1, first sentence, fits the various wishes of this thread succinctly, if 
not in a single word, and even not readily allowing an easy acronym.
Greetings,
John

Emeritus Professor John R Helliwell DSc



On 30 J

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Dear all,

While following the development of this thread, I am truly amazed how people 
cling to names for the number of measurements per reflection whose meaning:

  *   Depends on the cultural/engineering/scientific context
  *   Can only be understood by experts
  *   Where the experts, as witnessed by the discussions in this thread, do not 
agree on which name to use.

What is wrong with the name “measurements per reflection”? The definition for 
measurement is the same as is used to calculate the multiplicity/redundancy.
The only disadvantage I see is that it can be understood by non-experts as 
well, which reminds me of medical doctors, who invent complicated Latin names 
for common ailments to prevent patients to understand where they are talking 
about.

Another 2 cents/pennies from my side,
Herman



Von: CCP4 bulletin board  Im Auftrag von James Holton
Gesendet: Mittwoch, 1. Juli 2020 20:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk


Sorry to take this thread on a detour/diversion: What I was attempting to point 
out below, perhaps unclearly, is that the different interpretations of the word 
"redundant" are a cultural difference.  As a student of multiple English 
languages perhaps I can explain:

Few US English speakers know that in UK/European/Australian English the word 
"redundant" has a strong negative connotation. I, for one, was surprised to 
learn that the phrase "made redundant" is used in the UK to describe loss of 
employment.  That is, a layoff, firing or perhaps a furlough. So, I think it 
important to spell out for my fellow US English speakers that the emotional 
ties to this negative connotation can be strong ones.

Conversely, many UK English speakers do not know that in US English the word 
"redundant" has a strong positive connotation.  We never use the phrase "made 
redundant" to describe a lost job.  Most Americans I think would be confused by 
such a turn of phrase. If a US English speaker was told their jobs was "made 
redundant" they would most likely think that a new hire was onboarded to back 
them up.  This would imply that their job was so important that the company 
wanted at least two people doing it, just in case you got hit by a bus. This 
strong positive connotation also has emotional roots.

Personally, I prefer the positive connotation. Perhaps that is my cultural 
bias, or perhaps I just generally believe that positivity is better than 
negativity. Maybe I'm just a "nice" guy. The meaning of the word "nice" has 
changed enormously over the last few hundred years, and I don't think we're 
going to change that any more than we are going to change the meaning of 
"redundant" in these two major forms of English.

However, just because a word has slightly different meanings in two slightly 
different languages does not mean we should abandon it.  Are we going to stop 
eating "chips" just because we are not sure if our fried potato will come as 
sliced wedges or thin crispy wafers? If you are unhappy with your meal, is it 
the fault of the culture you are visiting? or the customer for forgetting where 
they are? Context is everything.

So, for those unfamiliar with one or more of the major English-speaking 
cultures, here are a few other important differences to be aware of:
"Football" may not be the game you think it is.
If you are offered a "biscuit" in the US, do not expect it to be sweet.
If you want to leave a building you should take the "lift" to the "ground 
floor", but if you take an "elevator" get off on the "1st floor".
A "dummy" is a pacifier for a baby in the UK/Australia, but in the US it only 
means an unintelligent person, or a plastic replica of one.
"please" and "thank you" are considered baseline politeness in some English 
cultures, but their excessive use in others, such as the US, can be seen as 
rude.
A "tap" in the US dispenses beer, water comes out of a "faucet".
A "flat" in the US is not a place to live, but rather where we test rocket cars.
"Gas" can be a liquid in the US.
"Rubber" is a substance in both languages, but in the US a lump of it meant for 
erasing pencil marks is an "eraser". Do not ask for a "rubber" at the shop 
unless you are sure which country you are in.
A "holiday" in the US is a special day on the calendar when everyone gets off 
work, not just when an individual takes a "vacation".
If you go walking down the "pavement" you are risking getting hit by a car in 
the US, because that is what we call the road bed, not the "sidewalk".
A "torch", is a handheld electric light in the UK, but in the US it is a 
flaming stick of wood.
A "queue" is a line of people in the UK, but in the US it is known only to 
computer scientists submitting jobs on a cluster.

Then there are words like "capillary", which means the same thing in both 
languages but the alternate pronunciations never 

[ccp4bb] AW: [EXTERNAL] [ccp4bb] flow rate for cooling stream?

[Schreuder, Herman /DE]

Dear Patrick,
if I recall correctly, our systems run at 10-15 ml/min (gas). I will check on 
Monday when I am back in the lab.
The original cryostreams would run for several day's on a tank of liquid 
nitrogen. However, they had significant hardware to dry the nitrogen and to 
ensure a constant flow.

Best, Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Patrick Loll
Gesendet: Donnerstag, 2. Juli 2020 22:03
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] flow rate for cooling stream?

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Sorry, way off topic:

Does anyone have an estimate for the flow rate one would typically use for the 
cold nitrogen stream passing over a protein crystal in a standard data 
collection?

Background: Our nitrogen “generator” has gone belly-up and the vendor no longer 
services it, so I’m testing the feasibility of using the boil-off from a liquid 
nitrogen tank to provide the gas to support a short data collection (this 
nitrogen gas would serve as the feedstock into our helium cryostat). But I 
don’t know the flow rate required, so I can’t calculate if one tank has enough 
nitrogen to support a day or so of data collection. There are flow meters for 
the warm and cold stream on the nitrogen generator, but these flow meters have 
no apparent units anywhere on them, so I have no idea of the rate at which the 
gas would be consumed.

Thanks for any useful tidbits. 

And for those of you in the US, best wishes for a happy “Holy crap, even MORE 
fireworks?!?!!” Day 

Pat


---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology Drexel University College of 
Medicine Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


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[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

nce a 
negative amount is meaningless, whereas if one couldn't care less the amount of 
caring must already be exactly zero, which is surely what the expression is 
meant to convey).  I'm not suggesting at all that I don't care, quite the 
opposite: I think it's vital that terminology is universally understood 
("define your terms, Sir, or we'll never agree").

So my 2p's worth is: carry on as we are, but please, please, please DEFINE (and 
only argue about the definitions!).

https://www.brainyquote.com/quotes/dylan_moran_557269?src=t_please_everyone<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.brainyquote.com_quotes_dylan-5Fmoran-5F557269-3Fsrc-3Dt-5Fplease-5Feveryone&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=rkv3VHbs8Bfme-xQfteNHrAgAmRdtVOsCD3ryGGQbTI&s=e3bL2Imbw_jrHBV5-UdMXDmTLVsgmIktLHjdQzF5Vjc&e=>

Cheers

-- Ian


On Thu, 2 Jul 2020 at 11:11, Harry Powell - CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk<mailto:193323b1e616-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Dear all

I’ve been persuaded that MPR is a useful name (and see that there are 
shortcomings with both “multiplicity” and “redundancy") and I agree with much 
of what’s been said most recently in this thread.

BTW, just because the Physics definition of a measurement/quantity/whatever is 
given on wikipedia (or elsewhere, for that matter), it doesn’t mean that’s what 
we (crystallographers, structural biologists, etc) should use without question. 
If you check


https://en.wikipedia.org/wiki/Reflection_(physics)<https://urldefense.proofpoint.com/v2/url?u=https-3A__en.wikipedia.org_wiki_Reflection-5F-28physics-29&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=rkv3VHbs8Bfme-xQfteNHrAgAmRdtVOsCD3ryGGQbTI&s=tj7dE-cxPqP4L8eRvjiu0CaZL3VNFn19dKa-9VTdVvM&e=>

you will find no mention of diffraction maxima corresponding to reflections 
except a link to a page on diffraction. Or maybe we should slavishly follow the 
Physicists and use another term…

H

> On 2 Jul 2020, at 10:41, Schreuder, Herman /DE 
> mailto:herman.schreu...@sanofi.com>> wrote:
>
> Dear all,
>
> While following the development of this thread, I am truly amazed how people 
> cling to names for the number of measurements per reflection whose meaning:
>   • Depends on the cultural/engineering/scientific context
>   • Can only be understood by experts
>   • Where the experts, as witnessed by the discussions in this thread, do 
> not agree on which name to use.
>
> What is wrong with the name “measurements per reflection”? The definition for 
> measurement is the same as is used to calculate the multiplicity/redundancy.
> The only disadvantage I see is that it can be understood by non-experts as 
> well, which reminds me of medical doctors, who invent complicated Latin names 
> for common ailments to prevent patients to understand where they are talking 
> about.
>
> Another 2 cents/pennies from my side,
> Herman
>
>
>
> Von: CCP4 bulletin board 
> mailto:CCP4BB@JISCMAIL.AC.UK>> Im Auftrag von James 
> Holton
> Gesendet: Mittwoch, 1. Juli 2020 20:52
> An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
> Betreff: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?
>
> EXTERNAL : Real sender is 
> owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>
>
>
>
>
> Sorry to take this thread on a detour/diversion: What I was attempting to 
> point out below, perhaps unclearly, is that the different interpretations of 
> the word "redundant" are a cultural difference.  As a student of multiple 
> English languages perhaps I can explain:
>
> Few US English speakers know that in UK/European/Australian English the word 
> "redundant" has a strong negative connotation. I, for one, was surprised to 
> learn that the phrase "made redundant" is used in the UK to describe loss of 
> employment.  That is, a layoff, firing or perhaps a furlough. So, I think it 
> important to spell out for my fellow US English speakers that the emotional 
> ties to this negative connotation can be strong ones.
>
> Conversely, many UK English speakers do not know that in US English the word 
> "redundant" has a strong positive connotation.  We never use the phrase "made 
> redundant" to describe a lost job.  Most Americans I think would be confused 
> by such a turn of phrase. If a US English speaker was told their jobs was 
> "made redundant" they would most likely think that a new hire was onboarded 
> to back them up.  This would imply that their job was so important that the 
> com

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Dear David,

Thank you for your reaction. It has become clear to me that although most 
people understand what I intended with “measurement”, in practice it is very 
much in the eye of the beholder. It was suggested in the BB to use observation 
instead, but I am fairly sure that some people will also have issues with that.

The advantage of multiplicity/redundancy is that it does not mention what is 
multiple or redundant and that one can refer to the program documentation for 
an exact definition. Since most people are happy with the 
multiplicity/redundancy they grew up with, that is the way it will stay.

Best regards,
Herman




Von: David Waterman 
Gesendet: Freitag, 3. Juli 2020 10:49
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames 
to get a full dataset?


EXTERNAL : Real sender is dgwater...@gmail.com<mailto:dgwater...@gmail.com>

Hi Herman,

I like the idea of MPR, but I continue to worry about the term "measurement". 
The intensity associated with a particular reflection is a fit based on a 
scaling model, and ultimately, depending on your integration software, may be 
linked to a weighted sum of two raw measurements: the summation and 
profile-fitted intensities. I think these are the measurements, not the 
intensity derived during the scaling procedure. Sure, anyone who wants to be 
even more pedantic than me will point out that these "raw measurements" are 
also the result of fitting procedures. However, to my eyes, the difference is 
that we don't consider the profile and summation integrated intensities to 
change as a result of the procedure that ultimately determines the statistic 
(MPR) of interest. During that procedure they are independent, not dependent 
variables.

Maybe I am worrying about nothing. It agree it is fairly clear what you mean by 
MPR. I just wanted to explore if there was any opportunity for further reducing 
ambiguity.

Cheers
-- David


On Fri, 3 Jul 2020 at 08:12, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Ian,

Since some very advanced countries still use miles, Fahrenheit and inches, I 
did not expect anything to change. It was an escalating discussion in this 
thread on data completeness(!) on the use of multiplicity vs redundancy that 
made me suggest a different term. Except for an occasional discussion in the 
BB, there is nothing against people using the term they are most comfortable 
with.

However, I insist that trying to impose a different definition of “measurement” 
for MPR vs the definition used for the calculation of redundancy/multiplicity 
is not a valid argument against MPR.

Cheers,
Herman




Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Ian Tickle
Gesendet: Donnerstag, 2. Juli 2020 22:06
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a 
full dataset?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>


Well I very much doubt that many software developers are going to trawl through 
all their code, comments, output statements & documentation to change 
'redundancy' or 'multiplicity' to 'MPR' or whatever terminology is agreed on 
(assuming of course we do manage to come to an agreement, which I doubt).  And 
good luck with persuading wwPDB to change 'redundancy' in their mmCIF 
dictionary!  That would be not only pointless but also a lot of work, partly 
because terms get abbreviated in code and in outputs (e.g. to 'redund' in mine, 
or 'mult').  And don't say I can keep the code & comments the same and only 
change the outputs and documentation: that will really tax my brain!  Also 
don't say this need only apply to new code: no code is ever completely new, and 
mixing up old & new terminology would be a disaster waiting to happen!  Also it 
won't end there: someone will always find terminology that they disagree with: 
I can think of plenty cans of worms that we could open, but I think one is 
already one too many!

By the way, "measurements per reflection" won't float, because some 
measurements will be rejected as outliers (that's why we need redundancy! - as 
opposed to simply measuring intensities for longer).  What I call redundancy is 
"the count of _contributing_ measurements per reflection" (CCMPR, sigh).  
Personally I think that adding one more term is going to confuse things even 
more since if I'm right most people will continue to use the old terms in 
parallel anyway.

IMO we should all be free to use the terminology we are most comfortable with, 
and it's up to the receivers of the information to perform the translation.  
That's how it always has been, and IMO always will be.  Of cou

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Schreuder, Herman /DE]

Hi David,

you are right, the M in MPR is just a count of “whatever” is averaged to get 
the final intensities.
However, from this “inexhaustible thread” it is also clear that there will be 
no agreement on what to call this “whatever” 😊

Best, Herman

Von: David Waterman 
Gesendet: Freitag, 3. Juli 2020 13:11
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames 
to get a full dataset?


EXTERNAL : Real sender is dgwater...@gmail.com<mailto:dgwater...@gmail.com>

Hi Herman,

I started googling and ended up completely lost down a rabbit hole (have a look 
here if you want to see what I mean: 
https://plato.stanford.edu/entries/measurement-science/<https://urldefense.proofpoint.com/v2/url?u=https-3A__plato.stanford.edu_entries_measurement-2Dscience_&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=NfK-XzbGoQVLv6T5t-KMc5bAwEQPdEr96sFwQ9Ep1Dc&s=mGF-gpMQiGiilkipkrQQ2T-lET7gg95-qT2CUIVe0gQ&e=>).
 As a result I'm no longer sure I know what the word "measurement" means! I 
tried to simplify things with a practical example. Let's say I take a set of 
clearly real world measurements (temperature values over time, say). I can take 
the mean of subsets of these values and maybe that is a "measurement" too - 
especially for readings taken in quick succession, expressly done to reduce 
measurement error. But what if I fit a line to a series of values, is the 
gradient of the line also a "measurement"? Maybe?

Anyway, for MPR it probably doesn't matter if the measurement is of a response 
variable, rather than something "raw". That's because MPR isn't actually 
affected by the values themselves (ignoring the thorny issue of outlier 
rejection), it is just a count of them.

Cheers
-- David


On Fri, 3 Jul 2020 at 11:22, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear David,

Thank you for your reaction. It has become clear to me that although most 
people understand what I intended with “measurement”, in practice it is very 
much in the eye of the beholder. It was suggested in the BB to use observation 
instead, but I am fairly sure that some people will also have issues with that.

The advantage of multiplicity/redundancy is that it does not mention what is 
multiple or redundant and that one can refer to the program documentation for 
an exact definition. Since most people are happy with the 
multiplicity/redundancy they grew up with, that is the way it will stay.

Best regards,
Herman




Von: David Waterman mailto:dgwater...@gmail.com>>
Gesendet: Freitag, 3. Juli 2020 10:49
An: Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>>
Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] number of frames 
to get a full dataset?


EXTERNAL : Real sender is dgwater...@gmail.com<mailto:dgwater...@gmail.com>

Hi Herman,

I like the idea of MPR, but I continue to worry about the term "measurement". 
The intensity associated with a particular reflection is a fit based on a 
scaling model, and ultimately, depending on your integration software, may be 
linked to a weighted sum of two raw measurements: the summation and 
profile-fitted intensities. I think these are the measurements, not the 
intensity derived during the scaling procedure. Sure, anyone who wants to be 
even more pedantic than me will point out that these "raw measurements" are 
also the result of fitting procedures. However, to my eyes, the difference is 
that we don't consider the profile and summation integrated intensities to 
change as a result of the procedure that ultimately determines the statistic 
(MPR) of interest. During that procedure they are independent, not dependent 
variables.

Maybe I am worrying about nothing. It agree it is fairly clear what you mean by 
MPR. I just wanted to explore if there was any opportunity for further reducing 
ambiguity.

Cheers
-- David


On Fri, 3 Jul 2020 at 08:12, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Ian,

Since some very advanced countries still use miles, Fahrenheit and inches, I 
did not expect anything to change. It was an escalating discussion in this 
thread on data completeness(!) on the use of multiplicity vs redundancy that 
made me suggest a different term. Except for an occasional discussion in the 
BB, there is nothing against people using the term they are most comfortable 
with.

However, I insist that trying to impose a different definition of “measurement” 
for MPR vs the definition used for the calculation of redundancy/multiplicity 
is not a valid argument against MPR.

Cheers,
Herman




Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Ian Tickle
Ge

[ccp4bb] AW: [ccp4bb] Accessing full list of programs in CCP4I2

[Schreuder, Herman /DE]

I fully agree, I have some old scripts I infrequently use. Making these 
programs inaccessible would break these scripts, forcing me to reinvent a 
couple of wheels.

My 2 cnts worth of junk to your mailbox,
Herman

Von: CCP4 bulletin board  Im Auftrag von Oganesyan, Vaheh
Gesendet: Mittwoch, 8. Juli 2020 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Accessing full list of programs in CCP4I2


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Thank you for explanation. There are many small programs in CCP4 that have not 
been updated for years and they still work despite being in unsupported folder. 
By making them inaccessible (or more difficult to access) nobody gets 
easier/cheaper life. Even if there are newer programs that may do the same it 
is not a good reason to forget the old ones. It is like forgetting the history 
and the culture. Then making new discoveries just to find that it is the same 
old “wheel” just a bit more round. It is probably very personal, but I do not 
throw away old edition of Thermodynamics text book when new one is coming out. 
And I’m not a hoarder, do not have OCD or ADHD.

This is already 4 cents together. Sorry to clutter your mailbox.

Vaheh (CCP4 user since 1992)

From: Christian Roth 
mailto:christianroth...@gmail.com>>
Sent: Tuesday, July 7, 2020 12:42 PM
To: Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>>
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Accessing full list of programs in CCP4I2

I understand that one is used to the programs one is familiar with and Eleanor 
especially is very fond of these things, but there are several things which can 
be done for example in Coot (symmetry coordinates etc) . It is just another way 
to do it. I had the chance and pleasure to work for a while with the 
programmers of some of these programs in one building and such decisions are 
not taken lightly. But one cannot support all programs and write for every 
program interfaces or even keep two interfaces alive. There are just not enough 
people and money to do that. Also most scientific programmers don't get paid to 
keep the things just running. Would it be better to have also programmers to 
keep legacy up to date and write and update GUI's, maybe? But again someone 
needs to pay for that.
Just my 2 cents.

Cheers
Christian


On Tue, Jul 7, 2020 at 6:30 PM Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:
… and how all these changes being justified?

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Eleanor Dodson
Sent: Tuesday, July 7, 2020 12:25 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Accessing full list of programs in CCP4I2

Yes - there are things I use all the time which are not part of the CCP4I2 list
pdbset to generate symmetry equivalents  or find the centre of mass etc etc
coordconv to turn orthogonal coordinates to fractional - after all we are 
crystallographers and need to relate models to unit cells..
distang to do a quick check on crystal contacts..
nd there must be more..
Eleanor

On Tue, 7 Jul 2020 at 17:16, Palm, Gottfried 
mailto:p...@uni-greifswald.de>> wrote:
For occasions like Laus, it would be useful to further on have access to the
CCP4 v7.0 Program Documentation,
even if the programs are not updated as Christian explained.
I was for instance looking for translating coordinates as in pdbset, but 
couldn't find a replacement in the ccp4i2 gui.
Greetings
  Gottfried


On Tuesday, 07-07-2020 at 18:00 Lau Kelvin wrote:
Ah I see! Great I can run it that way

And yes, Eleanor, after I realized that list was gone, I was panicking that 
there are some random things I used to do with those programs would no longer 
be possible. Good to know that the command line versions are still there in the 
7.1 distro.

Best,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 07.07.20, 15:51, "CCP4 bulletin board on behalf of Christian Roth" 
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of 
christianroth...@gmail.com> wrote:

yes Eleanor is right. command line still works.[:-)]
fft is also in 7.1 distribution.



On Tue, Jul 7, 2020 at 3:43 PM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Oh Lau - how I miss that list!
But if you just run fft online it is still distributed..wombat:Downloads 
eleanor$

fft hklin  mapout ..

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Quote source inquiry

[Schreuder, Herman /DE]

My guess is that the model no longer superimposed well onto the electron 
density map, which should be easy to spot.
Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Frances C. 
Bernstein
Gesendet: Donnerstag, 16. Juli 2020 13:44
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Quote source inquiry

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



I do not remember the date or the PDB entry but it was during the time that the 
PDB at BNL was including distance and angle outliers in the checking report 
sent back to the depositor.
It was a not-too-large protein and there were perhaps half a dozen outliers 
each on distances and angles which was typical of an entry without 'problems'.  
So I sent the proposed entry to the depositor and got a panicked call that 
something was wrong based on the depositor looking at a display of the entry.  
By the next day the depositor had figured out that s/he had decided to convert 
to orthogonal for deposition and mistyped one of the cell dimensions by 1 
Angstrom.  That length was about 135 so the error was less than 1% in one 
direction and I was very impressed that the depositor had spotted it 
graphically.

After I did the appropriate linear algebra to correct the coordinates I took a 
look at the distance and angle outliers.  Of course they were different but to 
my great surprise there were about the same number of outliers for the 'bad' 
and the corrected entries.  So based on the checking at that time we could not 
tell the bad from the good.

I would be interested to know what would happen now with all the additional 
checking that is available.  Perhaps someone should do that experiment.

  Frances

=
Bernstein + Sons
*   *   Information Systems Consultants
5 Brewster Lane, Bellport, NY 11713-2803
*   * ***
 *Frances C. Bernstein
   *   ***  f...@bernstein-plus-sons.com
  *** *
   *   *** 1-631-286-1339FAX: 1-631-286-1999
=

On Thu, 16 Jul 2020, Gerard DVD Kleywegt wrote:

> There was a case a few years ago (not too many though) where a 1.6 ? 
> structure had been solved using an incorrect value for the wavelength 
> (~5% too low, leading to a cell that was slightly too small for its 
> contents to be comfortable). It was later corrected so we could 
> compare their validation statistics. Some interesting observations:
>
> - the geometry had been very tightly restrained so that didn't give a 
> clue  about the cell error (WhatCheck only suggested a very small 
> change)
>
> - somewhat surprisingly (I thought) the Ramachandran plot did not 
> improve in  the correct model (0.3% outliers in the wwPDB validation 
> report), and the  sidechain rotamer outliers even got worse (from 1.5 
> to 2.5 %)
>
> - the map looked surprisingly good for the incorrect cell
>
> - however, RSR-Z told clearly that the map was not good enough for the 
> claimed  resolution - the model had 24% outliers! (3% in the corrected 
> model which  still only put it at the ~50th percentile)
>
> - another good indicator was the clashscore (went from 44 to 7)
>
> - the original model did not include an Rfree, but the R-value (>0.3 at 1.6?
>  resolution) ought to have provided a clue to the crystallographers 
> and  reviewers one would think
>
> It would be interesting to see what would happen if the wavelength 
> would be set 5% too high.
>
> --Gerard
>
>
>
> On Thu, 16 Jul 2020, Clemens Vonrhein wrote:
>
>> Hi Robbie,
>> 
>> On Wed, Jul 15, 2020 at 07:23:15PM +, Robbie Joosten wrote:
>>> At the same time if you have a a more relaxed approach to restraints 
>>> than you might find systematic deviations in bond lengths. A test 
>>> for that has been in WHAT_CHECK for decades and it actually works 
>>> surprisingly well to detect cell dimension problems.
>> 
>> Indeed.
>> 
>>> That said, the problem is uncommon now.
>> 
>> Not so sure about that: we all rely on an accurate value of the 
>> energy/wavelength from the instrument/beamline - and if that is off 
>> (for whatever reasons) it will result in incorrect cell dimensions 
>> and a systematic deviation from the various restraints.
>> 
>> This would even affect the best experiment done on the best crystal 
>> ... so fairly easy to spot at the refinement stage, especially if 
>> such an energy/wavelength offset is constant over a long period of 
>> time on a given instrument. To spot this at the data collection stage 
>> one would hope that at some point a crystal with very pronounced 
>> ice-rings will be looked at properly (and the fact these are not 
>> where we expect them to should cause some head-scratching).
>> 
>> Cheers
>> 
>> Clemens
>> 
>> #
>> ###
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>>

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Quote source inquiry

[Schreuder, Herman /DE]

Dear Jessica,

Thank you for this positive news on electron diffraction on small molecules. A 
point which is still not clear to me: is it possible to determine the absolute 
configuration with electron diffraction? Some claim that it cannot be done, 
others claim that it can be done using multiple diffraction events.

What is your experience?
Best, Herman

Von: CCP4 bulletin board  Im Auftrag von Jessica Bruhn
Gesendet: Samstag, 18. Juli 2020 02:16
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Quote source inquiry


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Hi Garib, Tim and James,

Thank you for the helpful information. I look forward to testing this out on 
some of our data. Hopefully it helps!

To Garib, I think that electron diffraction/microED of small molecules is 
actually in a pretty good position for this technique to really take off. To 
your concerns:
1. Our group is personally very happy with the data coming from our detector 
(CETA-D). The thicker scintillator really seems to have helped. And there are 
other good detectors out there. I have some data posted in zenodo in case you 
are interested (10.5281/zenodo.3905397 and 10.5281/zenodo.3937740).
2. Crystal handling is thankfully very straightforward for dry, small molecule 
crystals. Just dab a TEM grid on some (crystalline) powder and in most cases 
you should be ready to collect. Protein crystals are unfortunately 
significantly more difficult to work with. We'll see how work in that area 
progresses...
3. As for rotation, I am curious to hear what concerns you have about rotation? 
Are you concerned about completeness? Or the lower data quality in the high 
tilt angle frames? Or the accuracy of the goniometer with regard to position 
and constant speed maintenance? If your concerns are about completeness, I 
would say that we have been able to get fairly decent completeness by combining 
data from multiple crystals. In our hands (18 small molecule ED structures 
solved in house), about half of these reached >95% completeness, another 
quarter were >90% and the rest were in the 81-90% range.

You may also be interested to know that of these 18 small molecule structures, 
three had to be refined in REFMAC5 because the resolution was a little low 
(1.2-1.7A) or the data to parameter ratio was too poor for SHELXL. I understand 
your time is limited, but I do think that electron diffraction for small 
molecules is really gaining momentum. We have collected data from almost fifty 
different samples from our clients all across pharma since installing our new 
camera in September. Many of these probably won't end up in public databases, 
but they have been hugely impactful for these chemists.

Have a wonderful weekend.

Best wishes,
Jessica



On Fri, Jul 17, 2020 at 2:11 AM Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:
Dear Tim,


I understand the problem. If the problem is the distance only then only one 
parameter is needed for refinement of lattice parameters.

I do think that microED has good potential. However engineering problems need 
to be sorted out (detector, crystal handling, rotation etc).

When the problem becomes urgent then I can coniblue working on this problem. I 
have already implemented using all data (chemistry and crystal data) for 
lattice refinement. They need to be tested properly.
There are several issues that need to be sorted out.

Regards
Garib



On 17 Jul 2020, at 08:29, Tim Gruene 
mailto:tim.gru...@univie.ac.at>> wrote:

Dear Garib,

thank you very much for the details! If everything goes to plan, we are
going to use the Dectris QUADRO in September(ish), ideally also with
some protein crystals. In ED, distance calibration is more difficult
than with X-rays because of instabilities in the lens system (at least
with the older instruments), and because I do not work with a parallel
beam, but focus the beam onto the detector surface. This is not a very
reproducible process. In those cases where I got high resolution data,
the cell is often quite stable, and the distance can vary by about 5%...

Best regards,
Tim

On Thu, 16 Jul 2020 23:21:54 +0100
Garib Murshudov mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:


One correction: Model should after molecular replacement and few
cycles of refinement (perhaps with a little bir relaxed geometry, but
not too much).

There is an option to use a model after molecular replacement but it
is being migrated to another program that will have proper tests.

Regards
Garib



On 16 Jul 2020, at 22:34, Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>>
wrote:

Hi Tim,

There is an option to do unit cell parameter refinement (for all
six parameters in general which can only happen in P1). It is
undocumented.

Celrefine/lattice refine all # if you give scale instead of all
then only one parameter is refined.

Cellrefine select .  # use only atomic B value < Bmedian +
alpha * Binterquartile_range


The last command was add

[ccp4bb] AW: [EXTERNAL] chirality with electron diffraction

[Schreuder, Herman /DE]

Hi Tim,

thank you for your reply. The 1998 Schenk paper is "new" to me, I had seen this 
one: https://science.sciencemag.org/content/sci/364/6441/667.full.pdf
The background of my question was about the status in practice: Is it possible 
to routinely determine the absolute configuration of small molecules by 
electron diffraction, or is it something that in theory can be done, but only 
difficult in practice?

Best,
Herman



-Ursprüngliche Nachricht-
Von: Tim Gruene  
Gesendet: Montag, 20. Juli 2020 11:03
An: CCP4 bulletin board ; Schreuder, Herman /DE 

Betreff: [EXTERNAL] chirality with electron diffraction

EXTERNAL : Real sender is  tim.gru...@univie.ac.at   



Dear Herman,

The absolute configuration can be determined, although very differently from 
X-ray ccrystallography.

So far, two different experimental approaches have been published: 
Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890

and Brazda et al (2019), 10.1126/science.aaw2560 

The former is based on imaging, the latter is based on dynamical
refinement: Jansen, Tang, Zandbergen, Schenk (1998),
https://doi.org/10.1107/S0108767397010489

There is a very interesting paper by Burmester and  Schroeder Scanning 
Microscopy Vol. 11, 1997 (Pages 323-334). According to this paper, the 
anomalous signal in ED is actually stronger than in X-ray crystallography. As 
far as I know, this has not been pursued further.

Best wishes,
Tim

P.S.: This is a response to your email to Jessica Bruhns in the thread 'quote 
source inquiry'. This thread has reached an overflow, so I took the liberty to 
adjust the subject.

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University 
of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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[ccp4bb] AW: [EXTERNAL] chirality with electron diffraction

[Schreuder, Herman /DE]

Thank you Tim, I had the same impression. 
I am afraid that our chemists will have to wait a little longer before they 
routinely can get absolute configurations from electron diffraction.

Best Herman

-Ursprüngliche Nachricht-
Von: Tim Gruene  
Gesendet: Montag, 20. Juli 2020 14:21
An: Schreuder, Herman /DE 
Cc: CCP4 bulletin board 
Betreff: Re: [EXTERNAL] chirality with electron diffraction

EXTERNAL : Real sender is  tim.gru...@univie.ac.at   



Dear Herman,

no, I don't think this is routine, yet! 
First of all, instrument manufacturers need to catch up. What's on the market 
is quite behind what is possible - although, I don't have the instrument I'd 
love to have, but at least the meta data transfer to the image headers only 
works when you work with a native CCD camera (stone age, I know), not with a 
hybrid pixel detector.

Secondly, Lukas Palatinus' software is, as far as I know, not yet compatible 
with the rotation method, i.e. when you use XDS or DIALS or mosflm, etc, you 
cannot use his software for dynamic refinement. I also understand that dynamic 
refinement is quite time consuming. Lukas is working on this, though.

Best wishes,
Tim

On Mon, 20 Jul 2020 10:36:49 +0000
"Schreuder, Herman /DE"  wrote:

> Hi Tim,
> 
> thank you for your reply. The 1998 Schenk paper is "new" to me, I had 
> seen this one:
> https://science.sciencemag.org/content/sci/364/6441/667.full.pdf The 
> background of my question was about the status in practice: Is it 
> possible to routinely determine the absolute configuration of small 
> molecules by electron diffraction, or is it something that in theory 
> can be done, but only difficult in practice?
> 
> Best,
> Herman
> 
> 
> 
> -Ursprüngliche Nachricht-
> Von: Tim Gruene 
> Gesendet: Montag, 20. Juli 2020 11:03
> An: CCP4 bulletin board ; Schreuder, Herman /DE 
>  Betreff: [EXTERNAL] chirality with 
> electron diffraction
> 
> EXTERNAL : Real sender is  tim.gru...@univie.ac.at   
> 
> 
> 
> Dear Herman,
> 
> The absolute configuration can be determined, although very 
> differently from X-ray ccrystallography.
> 
> So far, two different experimental approaches have been published: 
> Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890
> 
> and Brazda et al (2019), 10.1126/science.aaw2560
> 
> The former is based on imaging, the latter is based on dynamical
> refinement: Jansen, Tang, Zandbergen, Schenk (1998),
> https://doi.org/10.1107/S0108767397010489
> 
> There is a very interesting paper by Burmester and  Schroeder Scanning 
> Microscopy Vol. 11, 1997 (Pages 323-334). According to this paper, the 
> anomalous signal in ED is actually stronger than in X-ray 
> crystallography. As far as I know, this has not been pursued further.
> 
> Best wishes,
> Tim
> 
> P.S.: This is a response to your email to Jessica Bruhns in the thread 
> 'quote source inquiry'. This thread has reached an overflow, so I took 
> the liberty to adjust the subject.
> 
> --
> --
> Tim Gruene
> Head of the Centre for X-ray Structure Analysis Faculty of Chemistry 
> University of Vienna
> 
> Phone: +43-1-4277-70202
> 
> GPG Key ID = A46BEE1A



--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University 
of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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[ccp4bb] AW: Modeling ATP/ADP

[Schreuder, Herman /DE]

Hi Reza,

Since nobody answered your question so far, I will do it now:
In our hands, ArpWARP autoligand is still the best program to automatically 
dock ligands into electron density maps. Alternatively, on could also try 
Rhofit from Global Phasing or some other docking program.

However, the results of even the best docking program are still inferior to the 
results an experienced crystallographer can achieve. As others said, here you 
will need all the information you can get hold of:

  1.  Superimpose homologous structures and look if the ATP position correlates 
with your electron density.
  2.  Fit the ATP in every possible pose, refine each one and look which one is 
the best.
  3.  Look if the binding mode makes sense, e.g. no highly charged phosphates 
in hydrophobic pockets.
  4.  Look if the bad electron density makes structurally sense, e.g. density 
disappearing after a freely rotatable bond, but not in rigid parts etc.
  5.  Use your scroll-wheel to scroll up and down the contour level of your 
electron density map: scrolling up may reveal the phosphate positions, since 
they should have the highest electron density; scrolling down may reveal parts 
of the molecule with low electron density due to low occupancy.
  6.  Keep in mind that you may have a mixture of ATP and ADP bound, causing 
disordered maps.

Good luck!
Herman

Von: CCP4 bulletin board  Im Auftrag von Reza Khayat
Gesendet: Donnerstag, 23. Juli 2020 18:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Modeling ATP/ADP


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk


Hi,



Can folks suggest programs for objectively docking ATP/ADP molecules into 
density? Our density is not so good, probably because of occupancy, and we'd 
like a less subjecting approach for modeling. Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Problems with refinement of nucleic acid structure

[Schreuder, Herman /DE]

Dear Rafal,

At this resolution, one sees many amino-acid side chains with alternative 
conformation, so it might be a good idea to test if this is also true for 
nucleotides.

Best, Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Rafal Dolot
Gesendet: Mittwoch, 29. Juli 2020 14:42
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Problems with refinement of nucleic acid structure

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear CCP4 users,

I'm working on the another nucleic acid duplex structure. After several stages 
of rebuilding and refinement I observed something like a "shadow" 
of the second duplex position. Have you any ideas to explain this phenomena? 
Should I place the second molecule with occupancy completed to the first one? 
Or maybe is it a kind of disorder in crystal lattice? 
Data were collected to 1.02A using synchrotron radiation (processed with XDS), 
but I have another datasets collected to lower resolution (from
Zn-SAD) with visible similar effects. Refinement was done using Refmac (R/Rfree 
21.5/24.0). Data not appeared to be twinned, but Xtriage detected 
pseudo-translational symmetry.

Thank you for any advices.

Rafal

|--|
|Rafal Dolot, Ph.D.|
|  |
|Polish Academy of Sciences|
|Centre of Molecular and Macromolecular Studies|
|Department of Bioorganic Chemistry|
|Macromolecular Crystallography Team   |
|Sienkiewicza 112  |
|90-363 Lodz, Poland   |
|Phone: +48(42)6803215 |
|Cell:  +48 502897781  |
|--|



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[ccp4bb] real real-space-refinement

[Schreuder, Herman /DE]

Dear BB,

I would like to do a real real-space-refinement of a protein against a cryo-EM 
map; not the mtz-based Refmac approach. A quick internet search produced a lot 
of Phenix hits, but little ccp4 hits. Does somebody know how to do this using 
ccp4 programs, or has someone a Coot script to do this?

Thank you for your help!
Herman



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

[Schreuder, Herman /DE]

Coot does it, but if one wants to do it for a complete protein, it is a lot of 
clicking. On the other hand, I have not tried to just select the N- and 
C-terminus for real-space refinement to see what happens. I would prefer to 
have some script to do it.
Best, Herman

Von: Eleanor Dodson 
Gesendet: Mittwoch, 29. Juli 2020 17:23
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement


EXTERNAL : Real sender is 
eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>

Wont coot do that?
Eleanor

On Wed, 29 Jul 2020 at 16:20, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear BB,

I would like to do a real real-space-refinement of a protein against a cryo-EM 
map; not the mtz-based Refmac approach. A quick internet search produced a lot 
of Phenix hits, but little ccp4 hits. Does somebody know how to do this using 
ccp4 programs, or has someone a Coot script to do this?

Thank you for your help!
Herman



To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

[Schreuder, Herman /DE]

Hi everybody,

Thank you very much for the many tips! I discovered that in Coot by clicking on 
the N- and C-terminus of a protein chain, one can indeed run a real-space 
refinement on the complete chain and the same is true for a rigid body fit. 
However, subsequent refinement with Refmac did not converge, so it looks like I 
still have to go manually through the chains. ☹
Still on my to do list are:
- test Isolde
- test Main
- test the wiggle-fit option in Coot.

Although the Refmac procedure consisting of converting the cryo-EM map into 
structure factors and running a "crystallographic" refinement on it seems to 
work, in my opinion the future will be a robust real-space refinement on the 
cryo-EM map itself, instead of a refinement on derived structure factors. 
However, this is my personal opinion.

Best regards,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Tom Burnley - 
UKRI STFC
Gesendet: Mittwoch, 29. Juli 2020 18:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Hi Herman,


There is lots of information on CCP-EM's modelling tools here:


https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ccpem.ac.uk_training_icknield-5F2019_icknield-5F2019.php&d=DwIFAg&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=aaxzRy8JT0AM0_MqNvU2fr6IB4wIbkY8SsgvQkYcw_w&s=D5VOMK2Fz8AOV1PCvJpD9tm914KUxJlsRNRWXYPb7tI&e=


This includes a tutorial on Refmac.  As you note this is done in reciprocal 
space but the CCP-EM pipeline handles conversions from MRC to MTZ format (and 
runs Refmac via CCP4).


All the best,


Tom


From: CCP4 bulletin board  on behalf of Boaz Shaanan 

Sent: 29 July 2020 16:31:46
To: ccp4bb
Subject: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

Hi,
Try searching ccp-em.
Boaz

Boaz Shaanan, Ph.D.
Department of Life Sciences
Ben Gurion University of the Negev
Beer Sheva
Israel

On Jul 29, 2020 18:29, "Schreuder, Herman /DE"  
wrote:
Coot does it, but if one wants to do it for a complete protein, it is a lot of 
clicking. On the other hand, I have not tried to just select the N- and 
C-terminus for real-space refinement to see what happens. I would prefer to 
have some script to do it.
Best, Herman

Von: Eleanor Dodson 
Gesendet: Mittwoch, 29. Juli 2020 17:23
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement


EXTERNAL : Real sender is 
eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>

Wont coot do that?
Eleanor

On Wed, 29 Jul 2020 at 16:20, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear BB,

I would like to do a real real-space-refinement of a protein against a cryo-EM 
map; not the mtz-based Refmac approach. A quick internet search produced a lot 
of Phenix hits, but little ccp4 hits. Does somebody know how to do this using 
ccp4 programs, or has someone a Coot script to do this?

Thank you for your help!
Herman



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[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

[Schreuder, Herman /DE]

Thank you. I think I will then first try to optimize the weight and then 
increase the cycles.
Herman

-Ursprüngliche Nachricht-
Von: Tom Burnley - UKRI STFC  
Gesendet: Donnerstag, 30. Juli 2020 14:35
An: ccp4bb@jiscmail.ac.uk; Schreuder, Herman /DE 
Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement

EXTERNAL : Real sender is  tom.burn...@stfc.ac.uk   



Hi Herman,

Just a couple of tips for Refmac...

1) increase number of cycles - we find some cases require a lot of cycles, more 
than you may be used from MX refinement.
2) optimise weight - this is important and at present the autoweighting option 
is not as reliable for EM refinement as MX.

You could run these in parallel whilst exploring the other very nice tools 
suggested 😉!

Cheers,
Tom





From: CCP4 bulletin board  on behalf of Schreuder, 
Herman /DE 

Sent: 30 July 2020 13:28:00

To: ccp4bb

Subject: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real 
real-space-refinement



Hi everybody,



Thank you very much for the many tips! I discovered that in Coot by clicking on 
the N- and C-terminus of a protein chain, one can indeed run a real-space 
refinement on the complete chain and the same is true for a rigid body fit. 
However, subsequent refinement with Refmac did not converge, so it looks like I 
still have to go manually through the chains. ☹

Still on my to do list are:

- test Isolde

- test Main

- test the wiggle-fit option in Coot.



Although the Refmac procedure consisting of converting the cryo-EM map into 
structure factors and running a "crystallographic" refinement on it seems to 
work, in my opinion the future will be a robust real-space refinement on the 
cryo-EM map itself, instead of a refinement on derived structure factors. 
However, this is my personal opinion.



Best regards,

Herman



-Ursprüngliche Nachricht-

Von: CCP4 bulletin board  Im Auftrag von Tom Burnley - 
UKRI STFC

Gesendet: Mittwoch, 29. Juli 2020 18:51

An: CCP4BB@JISCMAIL.AC.UK

Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement



EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk







Hi Herman,





There is lots of information on CCP-EM's modelling tools here:





https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ccpem.ac.uk_training_icknield-5F2019_icknield-5F2019.php&d=DwIFAg&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=aaxzRy8JT0AM0_MqNvU2fr6IB4wIbkY8SsgvQkYcw_w&s=D5VOMK2Fz8AOV1PCvJpD9tm914KUxJlsRNRWXYPb7tI&e=





This includes a tutorial on Refmac.  As you note this is done in reciprocal 
space but the CCP-EM pipeline handles conversions from MRC to MTZ format (and 
runs Refmac via CCP4).





All the best,





Tom





From: CCP4 bulletin board  on behalf of Boaz Shaanan 


Sent: 29 July 2020 16:31:46

To: ccp4bb

Subject: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] real real-space-refinement



Hi,

Try searching ccp-em.

Boaz



Boaz Shaanan, Ph.D.

Department of Life Sciences

Ben Gurion University of the Negev

Beer Sheva

Israel



On Jul 29, 2020 18:29, "Schreuder, Herman /DE"  
wrote:

Coot does it, but if one wants to do it for a complete protein, it is a lot of 
clicking. On the other hand, I have not tried to just select the N- and 
C-terminus for real-space refinement to see what happens. I would prefer to 
have some script to do it.

Best, Herman



Von: Eleanor Dodson 

Gesendet: Mittwoch, 29. Juli 2020 17:23

An: Schreuder, Herman /DE 

Cc: CCP4BB@JISCMAIL.AC.UK

Betreff: [EXTERNAL] Re: [ccp4bb] real real-space-refinement





EXTERNAL : Real sender is 
eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>



Wont coot do that?

Eleanor



On Wed, 29 Jul 2020 at 16:20, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear BB,



I would like to do a real real-space-refinement of a protein against a cryo-EM 
map; not the mtz-based Refmac approach. A quick internet search produced a lot 
of Phenix hits, but little ccp4 hits. Does somebody know how to do this using 
ccp4 programs, or has someone a Coot script to do this?



Thank you for your help!

Herman







To unsubscribe from the CCP4BB list, click the following link:

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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Problems with refinement of nucleic acid structure

[Schreuder, Herman /DE]

Dear Rafal,

Another few comments from my side:

1) I guess you tried to fit alternative positions for the bases but this did 
not work?
2) I also guess you refined already anisotropic B-factors?
3) from the figure you send, the absolute value of the difference density looks 
very low (maybe 10% of the average 2mFo-dFc density). This means that whereas 
some density for the bases shows up, you may not see very much for the rest of 
the molecule and the phosphates may not move.

If you have not yet done it, I would try to fit alternate positions and 
otherwise leave it as it is.

Best,
Herman
 

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Rafal Dolot
Gesendet: Donnerstag, 30. Juli 2020 14:06
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Problems with refinement of nucleic acid 
structure

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear all,

Thank you for the response. I will try to explain it more precisely.

The molecule of interest is a duplex with 9 nt length, but is paired on the 
length of eight bases, with overhangs at ends. Molecules form a parallel 
strings across the crystal lattice, parallel to C-axis, because of these 
stacked overhangs. The structure was solved by MR using Molrep. 
Trials using Phaser were failed. The initial model was obtained by ZN-SAD. 
Refinement was dome for space group P43212, with cell parameters
31.96 31.96 95.07 90 90 90, with one duplex molecule per AU.


Schreuder, Herman /DE wrote:

> At this resolution, one sees many amino-acid side chains with 
> alternative conformation, so it might be a good idea to test if this 
> is also true for nucleotides.

Dear Herman,
I'm working on some protein ultra-high resolution structures (around 1.0 A or 
higher), and alternative conformations are nicely visible on electron density 
maps. In this case, there is visible almost all molecule, when you switch 
contouring to 2 sigma or lower on Fo-Fc maps, so I think, in this case it's not 
the same situation.


Matthew Snee wrote:
> Maybe test the spacegroup with Zanuda, and reprocess with the most 
> likely lower symmetry group.
> I guess the stats should improve if you identify a pseudo symmetry 
> operator that is currently being treated as a true symmetry operator?

Eleanor Dodson wrote:
> Sometimes ghost like this mean there is a spacegroup error - absences 
> can be the result of the non-crystallographic translation and not be 
> truly indicitive of the spacegroup. What is the possible spacegroup 
> and what is the NC translation vector?

Dear Matthew and Eleanor,

I run Zanuda on my datasets, and the output (which is below) suggested, that 
spacegroup is right chosen.

Step 1.
R-factors for the starting model.
Transformation into a supergroup.
-
| Subgroup | Spacegroup | R.m.s.d. |   Refinement in tested group   |
|  || from the ||
|   Ref|| starting |  Rigid   | Restrained  |
|  || model, A |--|-|
|  ||  |R |R |  R-free  |
|--||--|--|--|--|
| >>  10   | P 43 21 2  |  0.0002  |--|  0.5107  |  0.4871  |
| 10   | P 43 21 2  |  0.0002  |--|--|--|
^

Step 2.
Refinements in subgroups.
There are 8 subgroups to test.
^
| >>  10   | P 43 21 2  |  0.0002  |--|  0.5107  |  0.4871  |
-
|  1   | P 1|  0.0883  |  0.5252  |  0.4985  |  0.4883  |
|  2   | C 1 2 1|  0.0828  |  0.5447  |  0.5006  |  0.4877  |
|  3   | P 1 21 1   |  0.0824  |  0.5367  |  0.5018  |  0.4921  |
|  4   | P 1 21 1   |  0.0789  |  0.5292  |  0.4971  |  0.4846  |
|  6   | P 21 21 21 |  0.0956  |  0.5380  |  0.5064  |  0.4929  |
|  7   | P 43   |  0.0935  |  0.5183  |  0.4952  |  0.4835  |
|  9   | C 2 2 21   |  0.0908  |  0.5435  |  0.5042  |  0.4910  |
| 10   | P 43 21 2  |  0.0855  |  0.5427  |  0.5097  |  0.4913  |
-
| <<   7   | P 43   |  0.0935  |  0.5183  |  0.4952  |  0.4835  |
^

Step 3.
Refinement of the best model.
Candidate symmetry elements are added one by one.
^
| >>   7   | P 43   |  0.0935  |  0.5183  |  0.4952  |  0.4835  |
---

[ccp4bb] AW: Going back to Coot 0.8

[Schreuder, Herman /DE]

Dear Paul,

Here I fully agree with Eike. With the real space refinement in the new coot 
the ligand often goes everywhere, except where it should go. Changing the Xray 
weight helps sometimes, but not always. In many cases I do not real-space 
refine and leave it to Buster to do the refinement. It would be very good if 
the old behavior could be reinstalled.

Best regards,
Herman

Von: CCP4 bulletin board  Im Auftrag von Schulz, 
Eike-Christian
Gesendet: Freitag, 4. September 2020 10:36
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Going back to Coot 0.8


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear Paul,

I have been working with coot for over 10 years now with little reason to 
complain.

However, in spite of trying for a few months now, I am not getting warm with 
coot 0.9.

I like the new eye-candy, and the more organized menus. But fitting residues 
and ligands into ED, has never before been so difficult, and frankly it annoys 
me that previously simple tasks have become an effort. It seems as if coot and 
I see different minima and we always disagree where to put the residue. At the 
moment all my data are at convenient resolutions of 1.7Å or better, so there is 
little ambiguity on that side.

I am using all default settings, but maybe there is something that needs to be 
changed?


  *   Is there a way to go back to the old (0.8-style) fitting functions in 
coot 0.9? If so how?
  *   If not, which of the last coot versions 
(https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/binaries/release/)
 would you recommend?

With best regards,

Eike






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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] AW: Going back to Coot 0.8

[Schreuder, Herman /DE]

Dear Paul,
Thank you for your explanation. I will try to adapt my way of working to the 
new coot.
Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Paul Emsley
Gesendet: Freitag, 4. September 2020 13:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Going back to Coot 0.8

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear Frustrated Coot 0.9 Users,

I'd like to let you know that you I have heard similar reports in the recent 
past (including, as you can see, from EJD). I have taken what I believe to be 
remedial action to address the most pressing issue.

I think the situation has arisen because Coot users are bringing previous 
experience of using RSR into their use of 0.9. Because the movement of the 
non-dragged atoms in 0.8.x was rather unsophisticated, many people learnt (I 
discovered, rather late in the day) to "flick" the dragged atom with a large 
and fast movement to get over a local energy barrier. (Needless to say, I 
hadn't intended for that to happen, the approved way to make such modifications 
was Ctrl-drag over-dragging.) Applying a large and fast flick to the dragged 
atom in
0.9 often leads to an undesired result. If one then misses that fact that an 
atom pull restraint is still in effect and goes on to drag on another atom, 
then confusion and frustration ensues.

For myself, by using eigen-flip and jiggle fit beforehand and then by combining 
pepflip, JED Flip and backrub rotamer during RSR I often don't even need to 
pull on the atoms. By adding in interactive contact dots, I can see what it is 
that's causing Coot to not move the atom to where I'm trying to drag it (the 
NBCs have been re-paramaterized and up-weighted in the 0.9.x rewrite). I have, 
from time to time in the past year, used Coot 0.8.x and to me it now feels 
painfully crippled. RSR in Coot 0.9.x is joyfully expeditious and pleasingly 
animated.

For now (which is to say, before Coot 0.9.1 is available) I suggest keeping an 
eye out for unsatisfied atom pull restraints, and using the "Clear Pull 
Restraints" at moments of confusion. Also, use less flick and more smoothness 
when dragging atoms. For example, a 180 degree rotation of the ribose from the 
Coot tutorial would be difficult, if not impossible using 0.8.x, but in 0.9.x 
one can pull on the hydrogen atom of the O5' 
to rotate it in a few seconds. I have added a (rather poor) video of me doing 
just that to my channel (I should make a better video).

With all that said, I am still listening - I will add a change shortly that 
will make the pull atom restraints more obvious by making them fatter, pinker 
and more opaque. If there are still problems and you could somehow make a 
screencast available to me that illustrates the problem, then I would be very 
interested to view it.

Regards,

Paul.


On 04/09/2020 10:05, Schreuder, Herman /DE wrote:
> Dear Paul,
> 
> Here I fully agree with Eike. With the real space refinement in the 
> new coot the ligand often goes everywhere, except where it should go. 
> Changing the Xray weight helps sometimes, but not always. In many 
> cases I do not real-space refine and leave it to Buster to do the refinement. 
> It would be very good if the old behavior could be reinstalled.
> 
> Best regards,
> 
> Herman
> 
> *Von:*CCP4 bulletin board  *Im Auftrag von 
> *Schulz, Eike-Christian
> *Gesendet:* Freitag, 4. September 2020 10:36
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [EXTERNAL] [ccp4bb] Going back to Coot 0.8
> 
> *EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk 
> <mailto:owner-ccp...@jiscmail.ac.uk>
> 
> Dear Paul,
> 
> I have been working with coot for over 10 years now with little reason to 
> complain.
> 
> However, in spite of trying for a few months now, I am not getting warm with 
> coot 0.9.
> 
> I like the new eye-candy, and the more organized menus. But fitting 
> residues and ligands into ED, has never before been so difficult, and 
> frankly it annoys me that previously simple tasks have become an 
> effort. It seems as if coot and I see different minima and we always disagree 
> where to put the residue. At the moment all my data are at convenient 
> resolutions of 1.7Å or better, so there is little ambiguity on that side.
> 
> I am using all default settings, but maybe there is something that needs to 
> be changed?
> 
>   * Is there a way to go back to the old (0.8-style) fitting functions in 
> coot 0.9? If so how?
>   * If not, which of the last coot versions
> 
> (https://urldefense.proofpoint.com/v2/url?u=https-3A__www2.mrc-2Dlmb.cam.ac.uk_personal_pemsley_coot_binaries_release_&d=DwIDaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=JMRzXNjcC-4YR8SFJTyrf_aNVcxumHrqFuPyV9

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] AW: Going back to Coot 0.8

[Schreuder, Herman /DE]

Dear Paul,

I fully agree with Georg and the others. As I said in my first reaction, with 
the new coot, the inhibitor or side chain often does not end up a little wrong, 
but goes off completely in the wrong direction, which makes me wonder whether I 
had somehow selected the wrong map. It might also be that the default settings 
that had been distributed with coot are wrong. I will try to find an example 
that is non-confidential, but often the moment you want something to go wrong, 
everything works perfectly. 

The old real-space refinement was intuitive and easy to use and did exactly 
what the user expected, without having to consult the manual!😊 The result might 
not have been perfect, but was good enough for subsequent Refmac, Buster, 
Phenix refinement.  

Best regards,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Georg Zocher
Gesendet: Mittwoch, 9. September 2020 17:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Going back to Coot 0.8

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear Paul,

people are complaining that they have issues fitting their model in real space 
using coot 0.9.x. Personally, I very briefly used coot other the last months 
but also have problems using RSR properly because I'm used to do it the way it 
was over the last 10 years. This might be due to my ignorance that I did not 
have a more closer look into the new features of RSR in 0.9 release and how to 
use them. But from the user site it's different to handle now.

I do not understand your Ferrari comparison (sorry) as I would not buy one even 
if I would have more money than one can spend. But I would take a cup of coffee 
as an example. If you always go to the same dealer to get your coffee in the 
morning as you know you get a very descent and excellent tasting cup of coffee 
that you fully enjoy every morning than you really get used to it. It might be 
than a hard time for you if your coffee dealer tells you: Sorry guys, I do have 
now an optimized coffee that is much better, taste a lot better, is fair 
produced,... It's just not the coffee you drunk over the last 10 years and I 
will not offer that one anymore. You might give the new one a chance, you might 
find it as excellent as the dealer but you might not. From my point of view 
it's the lack of choice that personally I do not like so much, especially if 
there is no other coffee dealer around...

Nevertheless, I'm aware and fully respect all the effort you put in the 
development of coot and I'm really grateful that coot is available.

All the best,
Georg



Am 08.09.20 um 17:44 schrieb Paul Emsley:
> On 08/09/2020 16:25, Georg Zocher wrote:
>>
>>
>> we have the same experience in our lab.
>
>
> What experience is that? I am still in the dark about you think is now 
> worse.
>
>
>> Personally, I did would not like to judge here, as so far, I did not 
>> have had enough time to get into the new RSR of coot 0.9.x by myself.
>> But many colleagues did not like the new refinement module maybe just 
>> as they are used to the method in all coot versions before.
>
>
> You have a Ferrari parked beside your house but you want to to take 
> the bus to work because that's what you've always done. Or maybe the 
> Ferrari is parked around the back and you don't know it's there?
>
>
>>
>> I just thought if it wouldn't be an option to let the user decide 
>> what kind of RSR implementation she/he would like to use and give 
>> them the choice via an option in coot preferences?
>
>
> That would be possible but not easy. Unlike much of the CCP4 suite, 
> Coot is Free Software. But, again... why would you want to take the 
> bus? Explain.
>
>
> regards,
>
>
> Paul.
>
> ##
> ##
>
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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin

[Schreuder, Herman /DE]

Dear Andy,

I few thoughts from my side, but no solution I am afraid:

  *   Your twinning operator -h, -k, l is the standard alternative indexing for 
P3x space group, which makes a lot of sense.
  *   P32 is a low symmetry space group, which makes MR easier, but this is 
offset by the NCS.
  *   In my hands, MR is surprisingly insensitive to twinning, so I would 
search for the molecules using the twinned data. Also, for MR one does not need 
extremely high resolution data.
  *   However, twinning and low resolution data seriously hamper "de novo" 
model building. So if you have good MR models for your d1 and d2 domains, you 
may have a good chance of solving the structure. If you would have to build 
them "de novo" in a MR electron density map, you may be doomed.
  *   What I would do, is to look at the phaser map using a very large map 
radius: say 35-40 Å or more and look in the solvent region if there are places 
with higher density that may suggest the presence of the missing domains. If 
something shows up, you can focus on those regions in your MR, or even try to 
manually fit the Ca chain of your MR model.
  *   You certainly have already done it, but Phaser has to option to search 
for additional domains, given the domains you already found.

Best,
Herman


Von: CCP4 bulletin board  Im Auftrag von Andrew Lovering
Gesendet: Montag, 14. September 2020 11:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Best protocols to advance a low resolution twin


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

To follow on from this thread:

To answer Jon, I did try to see if P6 subgroups were a possibility but can rule 
this out for a few reasons (MR doesn't give solutions, merging stats not 
suggestive of P6, the other dataset with the twin fraction that is 
significantly further from 50:50); and the d3:d3 NCS is not parallel to any 
crystallographic axis

The spacegroup is indeed P3 sub 2, not P321, and the solution again only 
possible in P3 sub 2 not P3 sub 1, so spacegroup confidence is high

I did get one reply from Petrus Zwart that twin refinement / map improvement is 
a subject being worked on

What I might try is a Phaser MR where the "missing domains" are searched for 
using cut out density of the one placed domain, rather than model (which could 
possibly be a better choice at this low resolution? Thoughts appreciated)

Best wishes & thanks everyone
Andy



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Can anyone hear me?

[Schreuder, Herman /DE]

In theory, one gets one’s own message as well. However, most spam filters, at 
least the one I got, block messages sent by the recipient.
Best, Herman

Von: CCP4 bulletin board  Im Auftrag von Eleanor Dodson
Gesendet: Montag, 21. September 2020 11:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Can anyone hear me?


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Well - I dont think one gets one;s own messages?
Eleanor

On Mon, 21 Sep 2020 at 10:07, Harry Powell - CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Hi Folks

Okay, you can stop replying to me now on this topic - I’ve had several replies 
from people on the BB telling me that they

(a) have received my message and/or
(b) have noticed that the BB is rather quiet at the moment

So it seems to be working - except that I haven’t even received my own message 
from the BB…

Harry

> On 21 Sep 2020, at 09:47, Harry Powell - CCP4BB 
> <193323b1e616-dmarc-requ...@jiscmail.ac.uk>
>  wrote:
>
> Hi folks
>
> SInce early last week I have had no messages from the BB - and also received 
> a message telling me that I had been unsubscribed automatically because of 
> bounced messages.
>
> I _should_ have now been resubscribed, but am not getting anything - and the 
> BB is no longer on JISCMAIL’s list of existing BBs.
>
> Is anyone else out there having issues?
>
> Harry
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] AW: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Schreuder, Herman /DE]

Hi Dhiraj,
you could also consider making a fusion protein of your protein with itself, 
with a suitable long linker (gly-ser-gly-ser etc.?) in between. At least that 
dimer won't dissociate.
Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Srivastava, 
Dhiraj
Gesendet: Mittwoch, 23. September 2020 21:39
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] dimeric tag to induce the homodimerization of 
protein


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Thanks everyone for all the nice suggestions.
regarding Matthew's question, Yes, There is a possibility of polydispersity due 
to the proteins making chain/aggregate, however the affinity between the 
dimeric protein A and monomeric protein B is poor and kinetics of dissociation 
is very fast thus complex can not survive gel filtration chromatography. So, by 
increasing the avidity, We are hoping that only 2:2 complex will survive the 
gel filtration and we will be able to isolate monodispersed complex for further 
biophysical studies like SAXS or crystallization.

Thank you
Dhiraj


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Gloria Borgstahl 
mailto:gborgst...@gmail.com>>
Sent: Tuesday, September 22, 2020 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

I was thinking the form of GFP that dimerizes.  This would also make it easy to 
track where the protein is.

On Tue, Sep 22, 2020 at 1:28 PM Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>> 
wrote:
Any dimeric tag should work if you add a long enough linker to satisfy your 
distance criterion.

GST, for example. Download the coordinates and get a rough idea how long the 
linker would have to be for your protein.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> wrote:

EXTERNAL MAIL
Hi
I want to make my protein dimeric to increase its affinity for its 
interaction partner which is a dimer. does anyone know a suitable tag/fusion 
protein which can be used as C terminal fusion for this purpose? I can not use 
any of the leucine zipper as I am looking for the distance between the c 
terminus to be around 30-40 A.


Thank you
Dhiraj


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UT Southwestern

Medical Center

The future of medicine, today.



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[ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

[Schreuder, Herman /DE]

A practice that was very popular before the Rfree came around was to fit a 
water molecule in every noise peak. One would get spectacular low Rfactors this 
way, but I cannot imagine that anyone would believe that this would be fitting 
and not over-fitting.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Sam Tang
Gesendet: Dienstag, 20. Oktober 2020 05:27
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] over-fitting? over-refinement?

Hi, the question may be a bit weird, but how do you define 'over-fitting' in 
the context of structure refinement? From users' perspective the practical 
aspect is to 'fit' the model into the density. So there comes this question 
from our juniors: fit is fit, how is a model over-fit?

BRS

Sam



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[ccp4bb] AW: Kevin Denkmann lädt Sie zur Zusammenarbeit auf 'Rechnungen' ein.

[Schreuder, Herman /DE]

Looks more like crowd-phishing to me! 😉
The original message did not make it through my spam filter and it may not be a 
good idea to open the shared file.
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Robbie Joosten
Gesendet: Dienstag, 20. Oktober 2020 16:16
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Kevin Denkmann lädt Sie zur Zusammenarbeit auf 
'Rechnungen' ein.

Working on your bills with the entire bulletin board. Is that crowdsourcing or 
what? 😉

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Kevin 
> Denkmann
> Sent: Tuesday, October 20, 2020 15:27
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Kevin Denkmann lädt Sie zur Zusammenarbeit auf 
> 'Rechnungen' ein.
> 
> 
> Kevin Denkmann shared a file with you
> 
> Here's the document that Kevin Denkmann shared with you.
> 
>  my.sharepoint.com:443/:b:/g/personal/kevin_denkmann_dunnlab_de/EZQT
> 7ED-bpdJtg5lG-H32GkByQoTQqzWyiKSIRel_DIrFA?e=4%3a9jNtyK&at=9>
>   Rechnungen
>   This link will work for anyone.
> Open  my.sharepoint.com:443/:b:/g/personal/kevin_denkmann_dunnlab_de/EZQT
> 7ED-bpdJtg5lG-H32GkByQoTQqzWyiKSIRel_DIrFA?e=4%3a9jNtyK&at=9>
> 
>   Privacy Statement  notifyp.svc.ms:443/api/v2/tracking/method/Click?mi=HgrSi7OfwUKiTn-
> 401hPpQ&tc=PrivacyStatement&cs=f97d4ae4336b3342c9a937ee3f36e84e&r
> u=https%3a%2f%2fprivacy.microsoft.com%2fprivacystatement%5c>
>   notifyp.svc.ms:443/api/v2/tracking/method/View?mi=HgrSi7OfwUKiTn-
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> 
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[ccp4bb] AW: [ccp4bb] ligand bound to only one chain in the crystal

[Schreuder, Herman /DE]

Dear Christian,
We occasionally observe binding to only one monomer of a multimeric complex. I 
don’t think this invalidates the biological significance of your finding. I 
would superimpose the different monomers to see if they have (slightly) 
different conformations that prevent ligand binding. Also for cocrystallization 
experiments, the presence of or absence of channels is irrelevant. This only 
matters for soaking experiments.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Christian 
GALICIA
Gesendet: Dienstag, 27. Oktober 2020 11:19
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] ligand bound to only one chain in the crystal

Hello,
In our structure only one chain in a crystallographic trimer (non-biological) 
shows a ligand bound to it (with clear density). There doesn't seem to be any 
channels (or lack of them) favoring that specific site. Can the community give 
your opinion on whether this can make the presence of the ligand or its 
biological role questionable, and give any examples of similar cases you might 
be aware of. Thank you.
--
Christian Galicia
Post Doctoral Scientist
E-mail: cgali...@vub.be





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[ccp4bb] AW: phenix.refine with ligand with ambiguous electron density

[Schreuder, Herman /DE]

Hi Nika,

Here you need some common sense. The green density in you polder map may just 
be the bulk solvent that was removed from the model to generate the polder map. 
In this case you have to use common sense and ask yourself a couple of 
questions:

  *   Is the density really from the ligand, of from some other component from 
your crystallization solution? Molecules like tris or hepes love it to 
masquerade for ligands of interest.
  *   Could it be that only part of the ligand is visible because the other 
parts are disordered or not present? This happens quite often. Instead of the 
full-ligand, a break-down product or reaction intermediate might have bound, or 
part of the ligand binding site is occupied by crystal contact.
In this case I would fit the part of the ligand which has convincing electron 
density, refine and look at the electron density maps to see if the fit to the 
density is convincing and if additional atoms could be added to the ligand. If 
you cannot fit the whole ligand, even after some efforts, I would submit a 
partially fitted model.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Nika Žibrat
Gesendet: Dienstag, 24. November 2020 12:29
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] phenix.refine with ligand with ambiguous electron density


Hello,



I have a question about protein-ligand, of which ligand displays an ambiguous 
electron density. I am solving a structure of protein with ligand  which was 
obtained via soaking. Structural characteristics indicate the ligand is present 
however the electron density is quite vague and too small for the size of the 
whole ligand. I did a Polder map which showed much larger area of green 
density. After insertion of my ligand into the green density in Polder I ran 
phenix.refine and there is a lot of red on the spot where the ligand is which 
was to be expected. This leaves me wondering how, if even do I incorporate the 
polder map data into my refine input.



My question is, how do I continue refining and validating the structure in this 
case?



Thank you,



Nika Žibrat




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[ccp4bb] AW: [ccp4bb] Crystal dissolved while checking under microscope

[Schreuder, Herman /DE]

Hi Prasun,

Apparently, when you just mixed the peptide solution with the reservoir 
solution, crystals formed. After equilibration, the crystals were gone.

1) what I would try: just mix peptide and reservoir solution, but do not 
equilibrate, e.g. do not use a reservoir solution. This is called batch 
crystallization.
Alternatively: Just after mixing, the pH will be somewhere between 5 and 6.5. 
After equilibration, it will be 6.5. Try reservoir solutions of different pH's, 
e.g. 6.0 and 5.5, but you might want to screen in finer steps.
2) I don't think you can use dissolved crystals for seeding.
3) The crystals you observed may also have been salt.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Prasun Kumar
Gesendet: Donnerstag, 26. November 2020 12:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Crystal dissolved while checking under microscope

Hi Group:

I have a peptide that got crystallized within 20-25 minutes after putting down 
the tray. 
The condition has 1.8M Ammonium Salt, 0.01 Cobalt (ii) Chloride hexahydrate and 
MES (0.1M) pH 6.5.
I dissolved my peptide in 25 mM Sodium Accetate (pH=5). 
PI of my peptide is 10.2
When I checked for the first time, to my surprise, I saw crystals in the above 
mentioned condition. After an hour upon checking the same drops, I was unable 
to find the crystals and I think they are dissolved. 

My questions are following:

1) Can we stop the crystals getting dissolved? I have put another tray at 4 
degree C, hoping that the crystals (if they appear) will be more stable.
2) Can I use the drop (in which I got crystals) for seeding?

Thank You
Prasun 



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[ccp4bb] AW: [ccp4bb] Finding partial occupancy monomer by MR ?

[Schreuder, Herman /DE]

Dear Phil,
0.32 is awfully close to 1/3, which brings a nice mathematical puzzle to my 
mind to see if the 1/3 occupancy is somehow related to the 3 fully occupied 
monomers... It may also be related to a (trigonal??) space group...

You probably have already tried it, but phaser has the option to give it 
already solved molecules and ask it to search for additional molecules. Here I 
would indeed lower the expected % homology significantly, to crudely compensate 
for the low occupancy. In contrast to the advice of Dale, I would play around 
with the % homology to find the value which works best.

My 2 cents,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Phil Jeffrey
Gesendet: Donnerstag, 10. Dezember 2020 14:49
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Finding partial occupancy monomer by MR ?

Preamble:
I have an interesting crystal form with 3 monomers (~400aa) at full occupancy 
and apparently one at much reduced occupancy.  It was built recently from 
Se-SAD and was in moderately good condition: Rfree=32% for trimer, 2.6 Å.  In 
recent refinement cycles it became obvious that there was a 4th monomer in a 
region of weaker/choppy 2Fo-Fc and Fo-Fc density that corresponded to a 
"confusing" set of low-occupancy SeMet sites found by SHELXD and Phaser-EP.  
The experimental map was bad in that region and was probably flattened during 
density modification anyway, in retrospect.

Question:
Phaser failed to find the 4th monomer after trivially finding the other
3 with a recent version of the monomer.  I'm wondering if there's a way to 
indicate "this one is partial occupancy" to Phaser, or if there's a way to 
improve the odds of success beyond just lowering the expected % homology.  Or 
if anyone has had success with other programs.  This is perhaps a rare edge 
case but I naively expected Phaser to work.

In the end I used the weak SeMet sites to locate the monomer and the occupancy 
appears to be around 0.32 in refinement.

Cheers,
Phil Jeffrey
Princeton



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[ccp4bb] AW: [ccp4bb] Problem in finding a MR solution

[Schreuder, Herman /DE]

Hi Suraj,

It is strange that the P3 crystals do not produce a MR solution. Since they all 
came from the same crystallization condition, you may want to check that no 
proteolytic cleavage of your protein has taken place and that your crystals 
only contain a fragment of your protein. You may also want to check that at 
least one complete molecule would fit into the asymmetric unit of your P3 
crystals.
Your R and Rfree values for your C2 data set may be artificially low due to the 
detwinning procedure, but as Eleanor mentions, they are not bad.

What I would do in any case is to rebuild and refine the structure of your C2 
crystals as well as possible. In the worst case that could be the only 
structure you get, but there are worse things. Most crystals structures in the 
PDB have been solved from a single crystal form. You can then use your rebuilt 
and refined model to run again MR with your P3 data. Maybe you are lucky and 
get a solution this time.

Best,
Herman


Von: CCP4 bulletin board  Im Auftrag von Eleanor Dodson
Gesendet: Freitag, 11. Dezember 2020 15:37
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Problem in finding a MR solution

Well - C2 is a sub cell for P3 so that isnt surprising, but a cell difference 
of 202 to 212 means it isnt isomorphous..
But an
Rw Rf/ of 26/31 isnt bad for such low resolution data?
Eleanor

On Fri, 11 Dec 2020 at 12:01, Suraj Kumar mandal 
mailto:tuoamicosu...@gmail.com>> wrote:
Dear Sir,

Yes, we have checked handedness also. We are using the same MR solution with 
other data.


With best regards
Suraj



On Fri, Dec 11, 2020 at 4:56 PM srajan kapoor 
mailto:kapoorsra...@gmail.com>> wrote:
Have you checked for handedness to solve this problem?.
You can also try to use the structure that you have solved for molecular 
Replacement of the other crystals.
I have only two things in mind currently. I hope it will work for you.

On Fri, Dec 11, 2020, 4:48 PM Suraj Kumar mandal 
mailto:tuoamicosu...@gmail.com>> wrote:
Dear All,

We are trying to solve a structure of a protein, for which we have collected 
five different home source data at 3.2-3.5 Ang resolution. We are processing 
the data using iMOSFLM and the program suggests P3 (and related) space groups 
for all the data. We are able to get a solution with one data (twinned) in C2 
space group with Rw/Rf of 26/28%. However, the same solution can not be 
obtained using other four data. Interestingly, one of these four data is not 
twinned. The only common thing in these four data, I find, is that they are 
crystallized in the same crystallization condition, whereas, the data which 
gives solution is crystallized in another condition.

The template has 69% sequence identity with the target protein.

Any suggestion to troubleshoot this would be appreciated.

With best regards,
Suraj

--
Suraj Kr. Mandal
Research Scholar
Structural and Computational Biology Lab
Department of Biosciences and Bioengineering
Indian Institute of Technology (IIT), Guwahati
Guwahati, Assam 781039
Ph.: 09678244566
E.mail: tuoamicosu...@gmail.com
   surajkrman...@iitg.ernet.in



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--
Suraj Kr. Mandal
Research Scholar
Structural and Computational Biology Lab
Department of Biosciences and Bioengineering
Indian Institute of Technology (IIT), Guwahati
Guwahati, Assam 781039
Ph.: 09678244566
E.mail: tuoamicosu...@gmail.com
   surajkrman...@iitg.ernet.in



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[ccp4bb] AW: [ccp4bb] NCS/Pseudo-symmetry

[Schreuder, Herman /DE]

Hi Bashir,
At the end of your log file, you get a warning message that you have 87% 
solvent, which is highly unlikely and a suggestion to search for more 
molecules. Did you try to search for more copies (say 4)?
Best Herman

Von: CCP4 bulletin board  Im Auftrag von Muhammad Bashir 
Khan
Gesendet: Donnerstag, 25. Februar 2021 21:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] NCS/Pseudo-symmetry


Hello everyone;



I collected data set which can be easily processed in a space grouop P21 (P1211)

at approx 3.7A. I procedeed with MR using phaser in phenix which give a 
solution with two copies.



** There were 8 solutions

** Pdb and/or Mtz files have been written with results for 1 of these solutions

** Solution #1 written to PDB file:  Khan_phaser.1.pdb
** Solution #1 written to MTZ file:  Khan_phaser.1.mtz
   Solution #1 annotation (history):
   SOLU SET  RFZ=3.4 TFZ=4.2 PAK=0 LLG=15 TFZ==5.5 RFZ=2.8 TFZ=27.6 PAK=5 
LLG=1173 TFZ==36.5 LLG=1210 TFZ==36.8 PAK=6
LLG=1210 TFZ==36.8
   SOLU SPAC P 1 2 1
   SOLU 6DIM ENSE ense_1 EULER7.6   86.43.3 FRAC  0.20  0.00  0.18 BFAC 
-1.90 #TFZ==5.5
   SOLU 6DIM ENSE ense_1 EULER7.6   86.43.2 FRAC  0.05  0.00  0.51 BFAC 
 1.60 #TFZ==36.8
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0444 #RMSD  1.29 #VRMS  1.64

   Solution #2 annotation (history):
   SOLU SET  RFZ=3.0 TFZ=5.5 PAK=0 LLG=10 RFZ=2.7 TFZ=25.6 PAK=0 LLG=1165 
TFZ==34.8 LLG=1198 TFZ==34.0 PAK=4 LLG=1198
TFZ==34.0
   SOLU SPAC P 1 2 1
   SOLU 6DIM ENSE ense_1 EULER   10.7   83.5  177.9 FRAC  0.24 -0.00  0.14 BFAC 
-3.15
   SOLU 6DIM ENSE ense_1 EULER  169.3   96.4  357.9 FRAC  0.91 -0.00  0.52 BFAC 
 1.82 #TFZ==34.0
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0705 #RMSD  1.29 #VRMS  1.65

   Solution #3 annotation (history):
   SOLU SET  RFZ=3.4 TFZ=4.1 PAK=1 LLG=-1 RFZ=2.8 TFZ=34.6 PAK=1 LLG=1146 
TFZ==39.7 LLG=1185 TFZ==39.2 PAK=3 LLG=1186
TFZ==39.2
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER8.2   86.42.0 FRAC  0.01 -0.00  0.01 BFAC 
-1.69
   SOLU 6DIM ENSE ense_1 EULER8.3   86.42.0 FRAC -0.14 -0.00  0.34 BFAC 
 1.72 #TFZ==39.2
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0406 #RMSD  1.29 #VRMS  1.64

   Solution #4 annotation (history):
   SOLU SET  RFZ=3.5 TFZ=5.0 PAK=6 LLG=13 RFZ=2.9 TFZ=30.5 PAK=6 LLG=1156 
TFZ==34.4 LLG=1183 TFZ==33.6 PAK=7 LLG=1183
TFZ==33.6
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER  359.5   82.02.7 FRAC -0.18 -0.00  0.47 BFAC 
-2.89
   SOLU 6DIM ENSE ense_1 EULER  359.5   82.12.8 FRAC -0.02 -0.00  0.14 BFAC 
 2.57 #TFZ==33.6
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0529 #RMSD  1.29 #VRMS  1.64

   Solution #5 annotation (history):
   SOLU SET  RFZ=3.5 TFZ=5.0 PAK=6 LLG=13 RFZ=3.0 TFZ=29.0 PAK=6 LLG=1115 
TFZ==34.1 LLG=1178 TFZ==33.6 PAK=7 LLG=1178
TFZ==33.6
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER  359.9   80.80.7 FRAC -0.18 -0.00  0.47 BFAC 
-1.57
   SOLU 6DIM ENSE ense_1 EULER  180.1   99.2  180.7 FRAC  0.34  0.50  0.20 BFAC 
 2.47 #TFZ==33.6
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0405 #RMSD  1.29 #VRMS  1.64

   Solution #6 annotation (history):
   SOLU SET  RFZ=3.5 TFZ=4.6 PAK=1 LLG=-0 RFZ=2.8 TFZ=36.2 PAK=1 LLG=1136 
TFZ==35.0 LLG=1169 TFZ==34.6 PAK=1 LLG=1169
TFZ==34.6
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER  359.2   83.01.5 FRAC  0.06  0.01  0.03 BFAC 
-2.58
   SOLU 6DIM ENSE ense_1 EULER  359.2   82.91.4 FRAC -0.09  0.01  0.36 BFAC 
 2.08 #TFZ==34.6
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0869 #RMSD  1.29 #VRMS  1.66

   Solution #7 annotation (history):
   SOLU SET  RFZ=3.5 TFZ=4.6 PAK=1 LLG=-0 RFZ=2.8 TFZ=35.3 PAK=1 LLG=1122 
TFZ==35.4 LLG=1149 TFZ==34.5 PAK=1 LLG=1148
TFZ==34.5
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER  359.2   82.52.4 FRAC  0.06  0.01  0.03 BFAC 
-1.93
   SOLU 6DIM ENSE ense_1 EULER  180.8   97.5  182.4 FRAC  0.78 -0.49  0.31 BFAC 
 2.44 #TFZ==34.5
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0809 #RMSD  1.29 #VRMS  1.65

   Solution #8 annotation (history):
   SOLU SET  RFZ=3.4 TFZ=4.1 PAK=1 LLG=-1 RFZ=2.8 TFZ=33.9 PAK=1 LLG=1083 
TFZ==38.5 LLG=1146 TFZ==38.5 PAK=4 LLG=1146
TFZ==38.5
   SOLU SPAC P 1 21 1
   SOLU 6DIM ENSE ense_1 EULER8.1   86.12.6 FRAC  0.01 -0.00  0.00 BFAC 
-1.77
   SOLU 6DIM ENSE ense_1 EULER8.1   86.22.6 FRAC  0.17 -0.00 -0.33 BFAC 
 1.66 #TFZ==38.5
   SOLU ENSEMBLE ense_1 VRMS DELTA +1.0762 #RMSD  1.29 #VRMS  1.65

CPU Time: 0 days 6 hrs 35 mins 53.63 secs (  23753.63 secs)
Finished: Tue Feb 16 23:27:36 2021

Got the following warnings:



WARNINGS


--
Warning: Intensity moments suggest possibility of twinning. Tests based on 
possible twin
laws will be more definitive.
--

--
Warning: The composition you have entered gives a solvent co

[ccp4bb] AW: [ccp4bb] Can twinning be seen in the diffraction pattern?

[Schreuder, Herman /DE]

Dear Marina,

A lot can happen with twinning andthe crystallization process does not always 
adhere to rules written in text books. 
The first thing I would do is to look at the predicted spots to see if the 
"split" spots are predicted as single spots (and would then really be split), 
or if they are predicted as two different spots (and would therefore not be 
split).
I assume that the cell dimensions of the P622 and P3(2)21 cell are the same (no 
doubling of some sell axis) and that the detection of twinning was based on the 
intensity distribution of the data set.
What may have happened is that if the transition between the two twin domains 
is not perfect, the two twin domains may have slightly different orientations, 
leading to split spots.

My 2 cents,
Herman 

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Marina Gárdonyi
Gesendet: Freitag, 12. März 2021 11:31
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Can twinning be seen in the diffraction pattern?


Hello everyone,

I am a PhD student at the Philipps-University in Marburg and I am currently 
writing my thesis.

I have problems to understand whether in my case twinning can be seen in the 
diffraction pattern or not.

I know that it depends on the type of twinning wheter you can see it in the 
diffraction pattern. The crystal had a resolution of 2.2 A.  
During processing it seemed to have the space group P622, but in the end it was 
P3(2)21. With phenix Xtriage I found out, that the data set was twinned. The 
twin law was -h,-k,l. So it should be merohedral twinning.
I read in a paper, that in case of merohedral twinning you cannot see it in the 
diffraction pattern. But in my case that seemed not to be the case because of 
splitted reflections. Or am I wrong???

I would be very happy to hear your opinion on that. Thanks in advance!

Best regards,
Marina

--
Marina Gárdonyi

PhD Student, Research Group Professor Dr. Klebe

Department of Pharmaceutical Chemistry

Philipps-University Marburg

Marbacher Weg 6, 35032 Marburg, Germany

Phone: +49 6421 28 21392

E-Mail: marina@pharmazie.uni-marburg.de

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[ccp4bb] crystallizing fusion proteins

[Schreuder, Herman /DE]

Dear Bulletin Board,
Sorry for the slightly off-topic question, but we are struggling with a 
receptor domain that expresses well as a fusion protein, but gets lost the 
moment it is cleaved from the fusion partner. It could be that the receptor 
domain is not or misfolded, but it could also be a solubility problem.

I have seen some crystal structures of fusion proteins with MBP and for 
membrane proteins, T4-lysozyme fusions are often crystallized. What is your 
experience? Would it be worth trying to express and crystallize a fusion 
protein, or would it be better to look for other constructs, e.g. to include 
more receptor domains?

Thank you very much for your advice!
Herman






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[ccp4bb] AW: [ccp4bb] High Rs

[Schreuder, Herman /DE]

Dear Sam,

The first thing that would come to my mind would be ice rings, but since you 
said you don't have them, there must be another reason for the high Rs.
Zanuda will give you back the space group you used for MR and maybe a higher 
symmetry space group, but the program cannot be used to confirm that the space 
group you assigned is correct. For that, you will have to run Phaser with all 
possible P2x2x2x permutations. I assume you have already done that. If not, 
that would be the second thing I would try. You could even use your rebuild 
model as a search model.

The next thing would be to look into the ligand you used. If the ligand has 
partial occupancy and induces some conformational changes in the protein, your 
crystal may contain proteins with a mixture of conformations, which may also 
give rise to higher Rfactors.

Good luck, Herman

Von: CCP4 bulletin board  Im Auftrag von Sam Tang
Gesendet: Donnerstag, 1. April 2021 14:28
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] High Rs

Dear all

I have a dataset processed to 2.2 A, P212121, with no major issues identified 
by Xtriage (no tNCS, no twinning, no ice ring, good completeness). Phaser-MR 
gave a good solution except some loop regions are shifted. There is only 1 
molecule in the ASU (and seemingly no more molecule can be accommodated). I 
fitted the displaced loops, and confirmed the space group by Zanuda, but the 
R-factors stuck at 0.34/0.41 range.  What other aspects should I look into? 
Twin-refinement? (I am a bit reluctant to do so because neither Xtriage nor 
Pointless report it is twinned) I should also point out that the crystal was 
grown with its ligand but I cannot see good density for it.

BRs

Sam



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[ccp4bb] AW: [ccp4bb] sugestions on weak diffracting protein crystals

[Schreuder, Herman /DE]

Dear Deepak,
the cryoprotection may destroy the diffraction. I would also try diffraction at 
room temperature, using special sleeves to prevent dehydration. With 20% 
PEG400, you could also try to freeze the crystals without using additional 
cryoprotectant. If you are lucky, it may work.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Deepak Deepak
Gesendet: Dienstag, 18. Mai 2021 12:08
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] sugestions on weak diffracting protein crystals

Dear all,

I have got multiple crystals (see picture 1) of a protein (8kDa) with a helical 
aromatic oligoamide foldamer (5kDa) but these crystals diffract very poorly 
(see the diffraction pattern in picture 2).

I prepare a 1.3mM:1.3mM complex of protein: foldamer in 20mM Tris, pH 7.5 
buffer. Crystals grew in 3-5 days in sitting and hanging drop at 20 Deg C and 
25 Deg C in the following conditions:

- 20% PEG 400, 0.1M MES pH 6.0
-20% PEG 400, 0.1M Sodium Cacodylate pH 6.0

Multiple cryo used were:
-25%Glycerol in mother solution
 -30% glycerol in water
-30%PEG 400,
-35% PEG 400
-20% PEG 8000 + 40% PEG 400 mix

Kindly suggest some methods/modifications on how can I improve the resolution 
and get better-diffracting crystals. Please let me know if you need more 
information.

Kind regards,
Deepak
Ph.D. Student

PS: The protein is a DNA binding protein and I have crystallized and solved the 
structure of this protein with its DNA partner and now I crystallized it with 
our foldamers but diffraction is not good. There are multiple structures of the 
Protein+DNA complex in literature but no apo-protein structure as the protein 
needs a binding partner to crystallize. We already have solution studies 
showing a good binding.



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[ccp4bb] AW: [ccp4bb] superpose crystal objects in CCP4MG or other software

[Schreuder, Herman /DE]

Dear Stefano,
I do not know if it is possible, but as a workaround, you could first expand 
your object by crystallographic symmetry and then do the superposition.
Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Stefano Trapani
Gesendet: Mittwoch, 2. Juni 2021 14:31
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] superpose crystal objects in CCP4MG or other software


Dear all

I would like to visually compare (using some molecular graphics software) the 
crystal packing of two different crystal forms of the same protein.

In order to identify the similarities/differences between the crystal packings, 
I need to change the default unit cell origin and orientation of one of the 
crystal forms.

  *   Is there a way, in CCP4MG or other molecular graphics software, to apply 
a given rotation/translation matrix to a "crystal object" (not to a single 
molecular object), so that further expansions of the object by  crystal 
symmetry will be consistent with the new origin/orientation ?

  *   If yes: can the transformation be defined by a least-squares 
superposition between molecules from the two crystal objects ?

Best regards

--
Stefano Trapani



Maître de Conférences

http://www.cbs.cnrs.fr/index.php/fr/personnel?PERS=Stefano%20Trapani

-

Centre de Biologie Structurale (CBS)

29 rue de Navacelles

34090 MONTPELLIER Cedex, France



Tel : +33 (0)4 67 41 77 29

Fax : +33 (0)4 67 41 79 13

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INSERM UMR 1054

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[ccp4bb] AW: Strange indexing problem

[Schreuder, Herman /DE]

Dear Rob,

Your screen shot shows a large empty region with difference density for the 
second half of the tetramer, so the my guess is that you have a tetramer in the 
asymmetric unit, but that your molrep program only found a dimer. Would a 
tetramer fit in the asymmetric unit?
If you have a tetramer in the asymmetric unit, there are several things you 
could do:

  1.  Construct a tetramer and use that as a search model.
  2.  Truncate surface loops, which may lead to rejection of valid solutions 
due to clashes.
  3.  Set the maximum allowed number of clashes higher to prevent rejection.
However, if only a dimer would fit in the asymmetric unit, you have a problem 
and you may want to search for statistical disorder in protein crystals.

Good luck,
Herman

Von: CCP4 bulletin board  Im Auftrag von Robert S 
Phillips
Gesendet: Montag, 5. Juli 2021 15:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Strange indexing problem

I collected data last week on crystals of tyrosine phenol-lyase obtained under 
new conditions.  The data have higher resolution than previous crystals, to 1.5 
A.  However, I can't get them to index in any space group but P1.  Usually, the 
space group is P21212.  The self-rotation function is attached.  The P1 data 
will give a molecular replacement solution, but it does not refine below 0.46.  
The P1 asymmetric unit fits a dimer, but the assembly is a tetramer.  In the 
map, I can see the difference peaks from the other dimer of the tetramer.  What 
could be causing this problem?

Rob

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net



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[ccp4bb] AW: Strange indexing problem

[Schreuder, Herman /DE]

Rob,

Wat is the Matthews number, would it fit with a very low percentage solvent, or 
would it not fit at all? What happens if you superimpose a tetramer on your 
dimer?

Best,
Herman

Von: Robert S Phillips 
Gesendet: Montag, 5. Juli 2021 16:32
An: Schreuder, Herman /DE 
Betreff: Re: Strange indexing problem

Herman:

That is the problem.  The unit cell in P1 will only accept a dimer, and the 
real asymmetric unit must be a tetramer.  I tried to double the dimension of 
the unit cell and reintegrate.  That gave acceptable statistics, but molecular 
replacement gave two separate dimers, not a tetramer, and it still did not 
refine.

Rob

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu<mailto:rsphill...@chem.uga.edu>
Web:  
http://tryptophan.net<https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net&data=04%7C01%7CHerman.Schreuder%40sanofi.com%7C480e122de54943e9cc0108d93fc1ac73%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C0%7C637610923344729892%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=aaZEYjjaLJctM7%2FpKbqBlute4JRdIfpQnXxl8MxTWyM%3D&reserved=0>
________
From: Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>>
Sent: Monday, July 5, 2021 10:25 AM
To: Robert S Phillips mailto:p...@uga.edu>>; 
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: AW: Strange indexing problem

[EXTERNAL SENDER - PROCEED CAUTIOUSLY]

Dear Rob,



Your screen shot shows a large empty region with difference density for the 
second half of the tetramer, so the my guess is that you have a tetramer in the 
asymmetric unit, but that your molrep program only found a dimer. Would a 
tetramer fit in the asymmetric unit?

If you have a tetramer in the asymmetric unit, there are several things you 
could do:

  1.  Construct a tetramer and use that as a search model.
  2.  Truncate surface loops, which may lead to rejection of valid solutions 
due to clashes.
  3.  Set the maximum allowed number of clashes higher to prevent rejection.

However, if only a dimer would fit in the asymmetric unit, you have a problem 
and you may want to search for statistical disorder in protein crystals.



Good luck,

Herman



Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Robert S Phillips
Gesendet: Montag, 5. Juli 2021 15:54
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] Strange indexing problem



I collected data last week on crystals of tyrosine phenol-lyase obtained under 
new conditions.  The data have higher resolution than previous crystals, to 1.5 
A.  However, I can't get them to index in any space group but P1.  Usually, the 
space group is P21212.  The self-rotation function is attached.  The P1 data 
will give a molecular replacement solution, but it does not refine below 0.46.  
The P1 asymmetric unit fits a dimer, but the assembly is a tetramer.  In the 
map, I can see the difference peaks from the other dimer of the tetramer.  What 
could be causing this problem?



Rob



Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology

University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu<mailto:rsphill...@chem.uga.edu>
Web:  
http://tryptophan.net<https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net&data=04%7C01%7CHerman.Schreuder%40sanofi.com%7C480e122de54943e9cc0108d93fc1ac73%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C0%7C637610923344729892%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=aaZEYjjaLJctM7%2FpKbqBlute4JRdIfpQnXxl8MxTWyM%3D&reserved=0>





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[ccp4bb] AW: [ccp4bb] Problems with crystals diffracting

[Schreuder, Herman /DE]

Hi Rakesh,

The first question I have is: why do the crystals dissolve after some time? Did 
you use a ligand for crystallization that might be turned-over by the protein?
What I would do first in your case is to try to find a stabilizing buffer. 
Since you have obtained nice-looking crystals, there is no need any more to 
stay at the solubility limit of the protein to allow crystal growth and you can 
move to a region in the phase-diagram where the protein is no longer soluble.

In practice, this would meant that if your crystals grow at say 25% PEG, you 
transfer them to a buffer with 30% or even 35% PEG. With a significantly higher 
precipitant concentration, chances are that your crystals will no longer 
dissolve. Also, the higher PEG or other precipitant concentration may pull some 
water from your crystals, resulting in a tighter crystal packing, similar to 
what is done with crystal dehydration devices like the free-mounter.

However, it may also be that your crystals are intrinsically disordered, but if 
you do not try it, you will never know.

Best regards,
Herman

Von: CCP4 bulletin board  Im Auftrag von Rakesh 
Chatterjee
Gesendet: Dienstag, 27. Juli 2021 17:55
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Problems with crystals diffracting

Hi everyone

I was working on a protein complex where one protein binds with the other 
protein  majorly based on its surface charge. The protein complex yields nice 
crystals but does not diffract. Additionally the  crystals used to dissolve 
within the drop when allowed for incubation both at 18 degrees and 4 degrees. 
Repeated rMMS microseeding, construct variations and changes in purification 
strategies did not yield any diffractions. Conditions involved for the protein 
complexes primarily involved  PEG with various strengths and pH variations. 
Looking for your suggestions and valuable ideas

Thanks in advance
Dr Rakesh Chatterjee
Umea University
Umea Sweden




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[ccp4bb] AW: [ccp4bb] how to fix helices-sheets getting converted to coil,

[Schreuder, Herman /DE]

Hi Firdous,
In general, display programs use the HELIX/SHEET records in the pdb and when 
these are missing, the programs generate the secondary structure themselves. If 
these HELIX/SHEET records are present for some part of the structure, but are 
missing for other parts, these other parts will show up as coil.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Firdous Tarique
Gesendet: Dienstag, 17. August 2021 14:24
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] how to fix helices-sheets getting converted to coil,

Hi

The representation of secondary structures have changed in my model. Helices 
and sheets are no longer appearing in the form it should be but appearing in 
the coiled conformation.

It all started when I splitted the 40S ribosome into two parts: head and body 
(In order to make head and body from the 40S subunit, I had deleted a few 
nucleotides and amino acids in the connecting region.), then I did rigid body 
fitting in chimera, then saved them separately with respect to map, then opened 
these coordinates in coot and joined them together (merged)  and finally ran 
real space refinement in Phenix.

Now when I am opening the refined pdb in Chimera, while the secondary 
structures are in their proper shape and form in the body, all have changed 
into coil-coil conformation in the head of 40S.

Is this a problem of Chimera or ChimeraX or something changed in the output of 
the refinement job due to which this is happening?.

Is there any option in Chimera or ChimeraX to fix it ? I remember in Pymol one 
can assign secondary structures if there is misrepresentation from the original 
structure.

Your suggestions and advice are appreciated.

Attached is the model in case someone wants to have a look.

Best

Firdous






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[ccp4bb] AW: [ccp4bb] malonate and histidine interaction

[Schreuder, Herman /DE]

Hi Ana,
If this histidine is part of the active site, you may want to look into the 
catalytic mechanism to see if this histidine could react with something like a 
malonate. Active site residues can do amazing things that normal residues 
cannot. If something from your protease inhibitor cocktail would have reacted, 
this should occur in non-malonate conditions as well.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Ana Ebrecht
Gesendet: Mittwoch, 18. August 2021 13:14
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] malonate and histidine interaction

Hello Jon,
Thanks for the comments. Yes, the His is part of the active site, and the 
distances are very close, like a covalent bond. That's why I was wondering if 
the malonate can be bound to the His.

We tried to model the malonate in the different conformation, but that didn't 
work. But I'll check about the protease inhibitor. Thanks!

Kind regards
Ana

On Tue, 17 Aug 2021 at 14:48, Jon Cooper 
mailto:jon.b.coo...@protonmail.com>> wrote:
Hello, only other thought was that malonate might be binding in two 
conformations, i.e. dual occupancy, and did you use a protease inhibitor 
cocktail, since the constituents can react? The density looks a bit like 
citrate, but too close to the His. I couldn't read the distances to the His in 
your figure. Is it part of the active site and if so can you say what the 
enzyme is? Anomalous difference maps are popular here, too. Is the surrounding 
structure totally OK because odd features in the difference map can indicate 
problems nearby e.g. I did see a leucine which looked slightly strained on the 
left, but maybe I am wrong. Good luck. Cheers, Jon.C.


Sent from ProtonMail mobile



 Original Message 
On 17 Aug 2021, 12:11, Ana Ebrecht < 
anaebre...@gmail.com> wrote:

Hi Jon,
Thanks for the reply. I don't think this is acetylation, because I only see 
this density in the crystals that grew with malonate. In other conditions 
doesn't show anything like that. So I thought it'd be the malonate. But I'll 
check on that.
Thanks for the suggestion.

Kind regards
Ana

On Mon, 16 Aug 2021 at 16:56, Jon Cooper 
mailto:jon.b.coo...@protonmail.com>> wrote:
Hello, could the His be partially acetylated?

Best wishes Jon.C.


Sent from ProtonMail mobile



 Original Message 
On 16 Aug 2021, 14:52, Ana Ebrecht < 
anaebre...@gmail.com> wrote:

Dear all,

I am building the structure of a protein that was crystallized in 0.2 M sodium 
malonate pH 5.0, 20% w/v polyethylene glycol 3,350.
During the refinement, we found what we think is a malonate molecule in the 
active site, but it seems like is bound somehow to the histidine (this His is 
the catalytic residue of the enzyme), almost like a covalent interaction. Under 
other conditions of crystallization, the protein bound a sulfate and an acetate 
in the site but did not show this type of interaction with the histidine.

We couldn't find anything that explains a reaction between the malonate and the 
histidine.
Does anyone have experience with this reaction or have seen something similar 
before?

Thanks
Kind regards
Ana





[cid:image001.jpg@01D79832.EF240160]



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[ccp4bb] AW: [ccp4bb] chain on 2-fold axis?

[Schreuder, Herman /DE]

Hi Peer,

Normally, if one defines some residues with an occupancy below 1.0, the 
nonbonding contact restraints with other residues are switched off. It is 
already some time ago, but if I recall correctly, I had similar problems that 
nonbonding contact restraints were not switched off for residues related by 
crystallographic symmetry if they were not exactly on a crystallographic axis. 
I don't know if this is the problem that you are facing, but here is what I did 
in the past:

For Xplor (and I guess the same is true for Phenix), one can explicitly switch 
on or off any interaction one likes, and did is what I did using Xplor. So you 
might want to have a look at Phenix. For Buster, I, or better, the global 
phasing people, created a gelly file to specifically exclude troublesome 
restraints.

Again, I don't know if this is your problem, but if so, I would look at Phenix 
or Buster to see if you can refine your structure with one of those programs.

Best,
Herman


Von: CCP4 bulletin board  Im Auftrag von Peer Mittl
Gesendet: Donnerstag, 26. August 2021 11:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] chain on 2-fold axis?

Der CCP4 community,

Is there a refinement program that can handle protein monomers sitting
on crystallographic 2-folds?

This is probably a strange question but we have the following situation.
We have a 2.6 Ang datasets in SG P3221 (Rpim=4%, Isa=19.6) and a clear
molrep solution with 2 chains, albeit with tNCS (0/0/0.5) that can be
refined to around 27/33% Rfactor. According to Vm a third chain could be
present. So far so good, but there is clear difference ED for a third
chain sitting exactly on the 2-fold. Since the protein has a peculiar
shape, one can tell even its orientation. I can relax the symmetry to
P32 (or even P1) and place the missing chain with 50% occupancy on the
2-fold. This model can be refined, but I do not like this work around,
because the data is clearly P3221.

Any hints on similar crystal pathologies and how they have been handled
would be helpful.

All the best,
Peer



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[ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

[Schreuder, Herman /DE]

Dear Peer and Eleanor,

This is indeed what I am suspecting: If the "twinning operator" in P32 puts 4 
out of 5 protein chains on top of symmetry mates, is the "true" space group 
then P32, with 5 twinned chains, or P3221 with 4 normal chains and 1 chain on a 
special position? I would vote for the latter.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Peer Mittl
Gesendet: Freitag, 27. August 2021 10:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Eleanor,

I indeed used r/tefmac for the refinement and it came up with the values
HKL (a=0.56), KH-L (a=0.44). It would be interesting to see if a
refinement in P3221 would come up with the same occupancies for the
alternative conformations for the "extra" chain on the 2-fold axis. It
seems as if the "well-ordered" chains (2 in P3221, 4 in P32) form a
sublattice with P3221 symmetry and it's just the "extra" chain, which
generates the twinning.

All the best,
Peer

On 26.08.2021 18:09, Eleanor Dodson wrote:
> Motto =mitti in predictive text!
>
> On Thu, 26 Aug 2021 at 16:52, Eleanor Dodson
>  >>
>  wrote:
>
> Great, motto. I think you have nailed it! Did you use tefmac for
> twinned refinement? And if so what did it suggest the twin
>  fraction is?
>
> On Thu, 26 Aug 2021 at 16:30, Peer Mittl mailto:mi...@bioc.uzh.ch%0b>> > wrote:
>
> Yes, the data indeed seems to be twinned and the tNCS has
> masked the twinning statistics, which is why I haven't
> considered it so far.
>
> I have not tried twinned refinement in C2 and P1 yet, but
> refining 4 chains in P32 with twinning yields a difference ED
> map that clearly indicates one (and just on!) orientation for
> the 5th chain. Thank you all for your suggestions.
>
> Have a nice evening,
> Peer
>
> -"CCP4 bulletin board" mailto:CCP4BB@JISCMAIL.AC.UK%0b>> > schrieb: 
-
> An: CCP4BB@JISCMAIL.AC.UK 
> 
> Von: "Kay Diederichs"
> Gesendet von: "CCP4 bulletin board"
> Datum: 26.08.2021 16:41
> Betreff: Re: [ccp4bb] chain on 2-fold axis?
>
> Dear Peer,
>
> I suspect that the true spacegroup has lower symmetry than
> P3221, and that there may be twinning masked by tNCS.
> Subgroups of P3221 are C2 and P32 (
> https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups
> >
> )
> and of course P1.
> What I'd do is process the data, and solve (use the best chain
> of the refined P3221 model for MR) and refine the structure in
> these spacegroups.
> Inspect the results: If P1 is clearly better than P32 and C2,
> P1 is correct.
> If C2 (P32) is clearly better than P32 (C2), then P1 should
> give the same R-values as the better one; if so, P1 can be
> discarded.
> Try this with and without twin refinement - although it's hard
> to compare R-values of non-twinned and twinned refinements.
>
> The automatic way to do this is with Zanuda. If you run that
> locally, you can make refmac do twin refinement.
>
> For all resulting structures, I'd also feed the resulting
> Fcalc (!) into pointless. That should reveal that the packing
> is indeed close to P3221.
>
> Best wishes,
> Kay
>
>
> On Thu, 26 Aug 2021 11:54:06 +0200, Peer Mittl
>  >>
>  wrote:
>
> >Der CCP4 community,
> >
> >Is there a refinement program that can handle protein
> monomers sitting
> >on crystallographic 2-folds?
> >
> >This is probably a strange question but we have the following
> situation.
> >We have a 2.6 Ang datasets in SG P3221 (Rpim=4%, Isa=19.6)
> and a clear
> >molrep solution with 2 chains, albeit with tNCS (0/0/0.5)
> that can be
> >refined to around 27/33% Rfactor. According to Vm a third
> chain could be
> >present. So far so good, but there is clear difference ED for
> a third
> >chain sitting exactly on the 2-fold. Since the protein has a
> peculiar
> >shape, one can tell even its orientation. I can relax the
> symmetry to
> >P32 (or even P1) and place the missing chain with 50%
> occupancy on the
> >2-fold. This model can be refined, but I do not like this
> work around,
> >because the data is clearly P3221.
> >
> >Any hints on similar crystal pathologies and how they have
> been handled
> >would be helpful.
> >
> >All the best,
> >Peer
> >
> >###

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

[Schreuder, Herman /DE]

Dear Lijun,
with this argument I agree: the interactions between the two orientations of 
the “twinned” chain and the neighboring molecules will be different and the 
interacting side chains will almost certainly have different orientations, 
which necessitates a twinning of the whole structure.
Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Lijun Liu
Gesendet: Freitag, 27. August 2021 14:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

I believe it is a twin from P32.



  1.  Not like the assignment of double conformations with partial occupancies 
to small part of asu, for examples, a side chain of lysin or a small fragment 
of a protein, which have both conformations stayed in the same specific asu at 
the same time.  For this p3221 asu, if one copy of that chain occupies the part 
of the asu, the other so-called conformation will not appear in the same asu at 
the same time, so the conformation and occupancy issue was arisen from 
different cell units, it is a twin from p32.
  2.  Talking about the asu of p3221, it holds 3 chains, the chain of interest 
assigned as 2 conformations related by a strict 2-fold.  Since you believe and 
have reduced to P3221, then you should restrain the two overall strictly (since 
absolutely most of these atoms are not on special positions) and not to refine 
the two 0.5 occupancies either, respecting the crystallographic 2-fold.  This 
is equivalent to perfect twin from p32 theoretically (although refinement with 
twin mode in p32 and none-twin mode in p3221 may give difference even large).  
If you refined and resulted in occupancies away from 0.5, the symmetry was 
proven to be broken, which supports P32 twinning.

Lijun

Sent from my iPhone


On Aug 27, 2021, at 6:56 AM, Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:

How P3221 can be an option if it assumes chain on axis? I guess I’m missing 
something, but per my belief only those sg will be possible for which there is 
no axis going through the extra molecule. P1 sg looks the only correct option 
here in my humble opinion.
Democracy (voting) depends on science. However, the reverse is not, thankfully.

Vaheh

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Peer Mittl
Sent: Friday, August 27, 2021 6:32 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Herman,

The answer probably depends on the impact of the "extra" chain on the
sublattice. If there is no impact the "true" space group is P3221 with
one chain on the special position. If the swapping of the extra chain
influences the sublattice P32 (or C2 or P1, as pointed out by Kay)
twinned to P3221 might be the better description.

All the best,
Peer

On 27.08.2021 10:56, Schreuder, Herman /DE wrote:
>
> Dear Peer and Eleanor,
>
> This is indeed what I am suspecting: If the “twinning operator” in P32
> puts 4 out of 5 protein chains on top of symmetry mates, is the “true”
> space group then P32, with 5 twinned chains, or P3221 with 4 normal
> chains and 1 chain on a special position? I would vote for the latter.
>
> Best,
>
> Herman
>
> *Von:* CCP4 bulletin board 
> mailto:CCP4BB@JISCMAIL.AC.UK>> *Im Auftrag von
> *Peer Mittl
> *Gesendet:* Freitag, 27. August 2021 10:17
> *An:* CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
> *Betreff:* Re: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?
>
> Dear Eleanor,
>
> I indeed used r/tefmac for the refinement and it came up with the values
> HKL (a=0.56), KH-L (a=0.44). It would be interesting to see if a
> refinement in P3221 would come up with the same occupancies for the
> alternative conformations for the "extra" chain on the 2-fold axis. It
> seems as if the "well-ordered" chains (2 in P3221, 4 in P32) form a
> sublattice with P3221 symmetry and it's just the "extra" chain, which
> generates the twinning.
>
> All the best,
> Peer
>
> On 26.08.2021 18:09, Eleanor Dodson wrote:
> > Motto =mitti in predictive text!
> >
> > On Thu, 26 Aug 2021 at 16:52, Eleanor Dodson
> > mailto:eleanor.dod...@york.ac.uk
<mailto:eleanor.dod...@york.ac.uk%20%3cmailto:eleanor.dod...@york.ac.uk%20%0b>> 
<mailto:eleanor.dod...@york.ac.uk%20%3cmailto:eleanor.dod...@york.ac.uk>>>
> wrote:
> >
> > Great, motto. I think you have nailed it! Did you use tefmac for
> > twinned refinement? And if so what did it suggest the twin
> >  fraction is?
> >
> > On Thu, 26 Aug 2021 at 16:30, Peer Mittl mailto:mi...@bioc.uzh.ch%0b>> <mailto:mi...@bioc.uzh.ch%0b>> 
<mailto:mi...@bioc.uzh.ch
<mailto:mi...@bioc.uzh.ch%20%0b>> <mailto:mi...@bioc.uzh.ch>>

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

[Schreuder, Herman /DE]

Dear Lijun and others,

As Kay rightly pointed out, twinning is fundamentally different from e.g. 
disorder or alternative conformations. However, it does not depend on a chain 
or side chain rapidly jumping between two states, but whether they are within 
coherent length of each other. Since most data are collected from frozen 
crystals, no side chain will be rapidly jumping between two states. They will 
be frozen either conformation A or conformation B and since these conformations 
are randomly distributed throughout the crystal, they are in general within 
coherent length of each other and causing interference of their diffracted 
X-ray’s, so their complex structure factors are added.

However, for twinned crystals, the twin domains (twin pieces as you call it), 
behave as independent crystals and their intensities are added and not their 
(complex) structure factors.

I did not realize it when I first looked at the thread but the correct 
treatment of these crystals will depend on whether there are twin domains 
present in the crystals, or whether the two orientations of the disordered 
chain are randomly distributed throughout the crystal. Given that the two 
orientations have different twin fractions, my bet is that the twin supporters 
are right.

Best,
Herman



Von: Lijun Liu 
Gesendet: Freitag, 27. August 2021 15:57
An: Schreuder, Herman /DE 
Cc: ccp4bb@jiscmail.ac.uk
Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Herman:

if you say “twinned” chains, then it already means same thing of the two and 
the side chain interactions could not be different (as you can see only one 
copy in the output coordinates), unless you talking about borders between twin 
pieces.

In this situation, the only I could imagine to be if P3221, it would have to 
make that chain very very rapidly jump between two states in the same asu, 
which can be easily proven wrong.

I do agree if refined under P3221 without restraining the two strictly, side 
chain interactions may show differences —— data always not perfect!

Lijun
Sent from my iPhone


On Aug 27, 2021, at 8:12 AM, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear Lijun,
with this argument I agree: the interactions between the two orientations of 
the “twinned” chain and the neighboring molecules will be different and the 
interacting side chains will almost certainly have different orientations, 
which necessitates a twinning of the whole structure.
Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Lijun Liu
Gesendet: Freitag, 27. August 2021 14:22
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

I believe it is a twin from P32.




  1.  Not like the assignment of double conformations with partial occupancies 
to small part of asu, for examples, a side chain of lysin or a small fragment 
of a protein, which have both conformations stayed in the same specific asu at 
the same time.  For this p3221 asu, if one copy of that chain occupies the part 
of the asu, the other so-called conformation will not appear in the same asu at 
the same time, so the conformation and occupancy issue was arisen from 
different cell units, it is a twin from p32.
  2.  Talking about the asu of p3221, it holds 3 chains, the chain of interest 
assigned as 2 conformations related by a strict 2-fold.  Since you believe and 
have reduced to P3221, then you should restrain the two overall strictly (since 
absolutely most of these atoms are not on special positions) and not to refine 
the two 0.5 occupancies either, respecting the crystallographic 2-fold.  This 
is equivalent to perfect twin from p32 theoretically (although refinement with 
twin mode in p32 and none-twin mode in p3221 may give difference even large).  
If you refined and resulted in occupancies away from 0.5, the symmetry was 
proven to be broken, which supports P32 twinning.

Lijun

Sent from my iPhone



On Aug 27, 2021, at 6:56 AM, Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:

How P3221 can be an option if it assumes chain on axis? I guess I’m missing 
something, but per my belief only those sg will be possible for which there is 
no axis going through the extra molecule. P1 sg looks the only correct option 
here in my humble opinion.
Democracy (voting) depends on science. However, the reverse is not, thankfully.

Vaheh

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Peer Mittl
Sent: Friday, August 27, 2021 6:32 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Herman,

The answer probably depends on the impact of the "extra" chain on the
sublattice. If there is no impact the "true" space group is P3221 with
one chain on the special pos

[ccp4bb] AW: [ccp4bb] A "funny" thing related to AlphaFold2 ....

[Schreuder, Herman /DE]

Dear Nick,

I had just looked at a pdb downloaded from the alphafold server without 
problems. However, then I realized that I had looked at the alphafold model 
after I had it superimposed on my own structure. Loading the alphafoldmodel 
directly in coot failed for me as well.

By looking into the pdb file, I discovered that the alphafold file has a 
"MODEL" record just before the coordinates and an "ENDMDL" record after the 
coordinates. After deleting these two records, the alphafold pdb loads fine.

Hope this helps,
Herman

Von: CCP4 bulletin board  Im Auftrag von Nicholas Keep
Gesendet: Freitag, 27. August 2021 16:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] A "funny" thing related to AlphaFold2 

Has anyone made use of an Alpha Fold PDB as opposed to CIF.  On the half
dozen or so I have tried to read into CCP4mg or coot the PDB has always
failed but the CIF is fine.  I can then write out a PDB if I want.

I could add to the conspiracy theories that this is EBI trying to
normalise use of CIF format, but I suspect it is something much more
mundane, that should be addressed.

Best wishes

Nick

--
NOTE NEW PHONE NUMBER JULY 2020

I do not work Mondays. For urgent business on a Monday contact Clare Woodward 
(Director of Operations) or Gillian Forrester (Deputy Dean)

Prof Nicholas H. Keep
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Crystallography, Institute for Structural and Molecular Biology,
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office)

If you want to access me in person you have to come to the crystallography 
entrance
and ring me or the department office from the internal phone by the door



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[ccp4bb] AW: Antwort: [ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

[Schreuder, Herman /DE]

Dear Peer,

I must admit that from the beginning I was not happy to have 5 chains 
“twinned”, while in fact only one chain is “twinned”, e.g. on a special 
position. However, a twin fraction different from 0.5 suggested the existence 
of twin domains and not a random distribution of A and B conformations.

On the other hand, given that only 20% of the asymmetric unit is twinned, with 
tNCS present and imperfect experimental data, the twin fraction obtained might 
not be very accurate and a twin fraction of 0.5 could be within the error 
margin. The low Rfactors you obtain without twinning also point into this 
direction.

What I would do is have a critical look at the crystal packing to see if there 
are reasons why larger twin domains with the rogue chain only in one 
conformation would form. E.g. are there direct crystallographic contacts 
between these disordered that may force the disordered chain next to a chain in 
the “A” conformation to adopt the A conformation as well. Or is the disordered 
chain enclosed in the 4 non-disordered chains, with only crystal contacts 
between the non-disordered chains? If there is no compelling reason why 
neighboring disordered chains would adopt the same conformation, I would assume 
statistical disorder and not twinning.

My 2 cts,
Herman

Von: mi...@bioc.uzh.ch 
Gesendet: Dienstag, 31. August 2021 17:46
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Antwort: [ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] 
chain on 2-fold axis?

Dear Herman, Kay, Eleanor and CCp4C

At the beginning I was very enthusiastic about the twinning option, but 
meanwhile the enthusiasm has vanished. I basically get the same Rfactors after 
refining the structure in P32 with 5 chains in Refmac (test reflections 
selected in resolution shells with sftools straight from the beginning).

Refinement with tight NCS restrains on 4 chains and twinning results in 
Rf/Rfree 24.2/30.0, without twinning I get 27.0/32.5. With slightly relaxed NCS 
restrains for the side chains and no twinning its 24.3/31.8. A Rfree difference 
of 2% is that sufficient to confirm twinning?

What bothers me even more is the difference ED for the "extra" chain (the on on 
the special position in P3221). After refienement with twinning the difference 
density is slightly reduced but it is still present. Furthermore, I can replace 
the "extra" chain by a 180° rotated copy and the refinemnt ends up in basically 
the same Rfactors. I attached two screen shots from the superposition of the 
initial structure (all atoms in wheat) on the rotated structure (blue Ca-trace) 
with the corresponding ED maps.

All the best,
Peer

-"CCP4 bulletin board" 
mailto:CCP4BB@JISCMAIL.AC.UK>> schrieb: -
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Von: "Schreuder, Herman /DE"
Gesendet von: "CCP4 bulletin board"
Datum: 27.08.2021 16:37
Betreff: [ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 
2-fold axis?


Dear Lijun and others,

As Kay rightly pointed out, twinning is fundamentally different from e.g. 
disorder or alternative conformations. However, it does not depend on a chain 
or side chain rapidly jumping between two states, but whether they are within 
coherent length of each other. Since most data are collected from frozen 
crystals, no side chain will be rapidly jumping between two states. They will 
be frozen either conformation A or conformation B and since these conformations 
are randomly distributed throughout the crystal, they are in general within 
coherent length of each other and causing interference of their diffracted 
X-ray’s, so their complex structure factors are added.

However, for twinned crystals, the twin domains (twin pieces as you call it), 
behave as independent crystals and their intensities are added and not their 
(complex) structure factors.

I did not realize it when I first looked at the thread but the correct 
treatment of these crystals will depend on whether there are twin domains 
present in the crystals, or whether the two orientations of the disordered 
chain are randomly distributed throughout the crystal. Given that the two 
orientations have different twin fractions, my bet is that the twin supporters 
are right.

Best,
Herman





Von: Lijun Liu mailto:lijunli...@gmail.com>>
Gesendet: Freitag, 27. August 2021 15:57
An: Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>>
Cc: ccp4bb@jiscmail.ac.uk<mailto:ccp4bb@jiscmail.ac.uk>
Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis?

Dear Herman:



if you say “twinned” chains, then it already means same thing of the two and 
the side chain interactions could not be different (as you can see only one 
copy in the output coordinates), unless you talking about borders between twin 
pieces.



In this situation, the only I could imagine to be if P3221, it would have to 
make tha

[ccp4bb] AW: [ccp4bb] Phosphatase enzymatic assay

[Schreuder, Herman /DE]

It may also be possible to separate the different phosphorylated forms on an 
isoelectric focusing gel.
Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Patrick Loll
Gesendet: Mittwoch, 15. September 2021 15:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Phosphatase enzymatic assay

Phos-tag gels are an option (albeit a fiddly one).
Cheers,
Pat Loll


___

Patrick J.  Loll, PhD (he, him, his)
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(267) 359-2653
pj...@drexel.edu



On 15 Sep 2021, at 8:28 AM, Andrea Moretti 
mailto:andrea.more...@unige.ch>> wrote:

Dear CCP4 community,

sorry for the off topic question but I am struggling with enzymatic assays and 
I would need your help.

I am trying to measure dephosphorylation of a phosphorylated kinase by a 
ser/thr phosphatase. So far I tried to measure it by malachite green assay, 
anti pSer/pThr antibody and PNPase coupled assay but nothing really worked for 
me. NMR is unfortunately not an option since I can't make enough of the 
proteins.

So any hints on phosphatase assays would be helpful.
Thanks!

Best wishes,
Andrea


--
Andrea Moretti
Structural Plant Biology Laboratory
Department of Botany and Plant Biology
Sciences III
University of Geneva
30 Quai E. Ansermet
1211 Geneva
Switzerland
Email: andrea.more...@unige.ch
Phone: +41 22 379 3029 (office)
web:http://structplantbio.org



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[ccp4bb] AW: [ccp4bb] Protein's C-terminal neutral

[Schreuder, Herman /DE]

Dear Rohit,

Do you mean that the residues after your "C-terminus" have been deleted because 
there is no convincing electron density for them?
In that case, a charged carboxylate at the N-terminus is incorrect and you 
should delete the OXT atom. Alternatively, you could add one residue at the 
C-terminus and delete all atoms except the N. however, you may still have to 
manually delete the OXT atom.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von rohit kumar
Gesendet: Mittwoch, 22. September 2021 23:29
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Protein's C-terminal neutral

Hello Everyone,

Is there any way that I could make my protein's C-terminal neutral using Coot?
Actually, I have a protein-peptide complex structure and my peptide is bound at 
the C-terminal end. While making a surface charge diagram it is negatively 
charged (because of the CO group at the end, which is not the end residue of my 
purified protein)  and I want to make it neutral (possibly a peptide bond in 
solution).

Please let me know if I am clear enough with my question.

Thank you

--
Regards
Dr. Rohit Kumar Singh
Postdoctoral fellow
Aurora CO USA





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[ccp4bb] AW: [ccp4bb] what would be the best metric to asses the quality of a mtz file?

[Schreuder, Herman /DE]

Dear Murpholino,

What was the reason of trying all these different processing methods? I, and I 
guess most other crystallographers will process the data using a standard 
procedure and if the results are good, will not try a myriad of other 
processing methods. If it is to get most out of a poorly diffracting crystal, I 
would go for the data set with the best resolution, completeness and 
statistics. If it is to better see a poorly defined ligand, you have to be 
careful. You may be selecting the data set who's noise best resembles the 
ligand, which you dearly want to see, but which is not there. The twilight 
server provides lots of examples of what will happen then. So also here, I 
would select the data set based on resolution, completeness and statistics. 
Alternatively, you could see which data set gives the best Rfree after a few 
rounds of refinement.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Murpholino 
Peligro
Gesendet: Mittwoch, 27. Oktober 2021 19:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] what would be the best metric to asses the quality of a 
mtz file?

So... how can I get a metric for noise in electron density maps?
First thing that occurred to me
open in coot and do validate->difference map peaks-> get number of peaks
(is this scriptable?)
or
Second
phenix.real_space_correlation detail=residue file.pdb file.mtz


Thanks again

El mié, 27 de oct. de 2021 a la(s) 10:52, vincent Chaptal 
(vincent.chap...@ibcp.fr) escribió:
Hi,

It's hard to find a single metric...
Ultimately, the quality of electron density maps, lower noise in fo-fc?

Best
Vincent
Le 27/10/2021 à 17:44, Murpholino Peligro a écrit :
Let's say I ran autoproc with different combinations of options for a specific 
dataset, producing dozens of different (but not so different) mtz files...
Then I ran phenix.refine with the same options for the same structure but with 
all my mtz zoo
What would be the best metric to say "hey this combo works the best!"?
R-free?
Thanks

M. Peligro



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--

Vincent Chaptal, PhD

Director of GdR APPICOM

Drug Resistance and Membrane Proteins Lab



MMSB -UMR5086

7 passage du Vercors

69007 LYON

FRANCE

+33 4 37 65 29 01

http://www.appicom.cnrs.fr

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[ccp4bb] MrBUMP for cryo-EM

[Schreuder, Herman /DE]

Dear Community,

I just came accross a paper on the use of MrBUMP to fit pdb models to cryo-EM 
maps: https://scripts.iucr.org/cgi-bin/paper?S2059798321009165
However, it turned out that the program tries to download coordinates directly 
from the pdb, which our firewall does not allow. Is there a way to point MrBUMP 
to an internal mirror of the pdb?

Thank you for your help,
Herman




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[ccp4bb] AW: [ccp4bb] MrBUMP for cryo-EM

[Schreuder, Herman /DE]

Hi Adam,

Thank you for your response. Using the pdblocal keyword, I was able to run 
MrBUMP. Also the cryo keyword was recognized, but then the mode of operation 
was set to "MODELS" and only search models were generated. From your email, I 
understand that I will have to wait for ccp4 update 017 next week in order to 
be able to run molecular replacement.

Is this correct?

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Adam Simpkin
Gesendet: Freitag, 5. November 2021 12:45
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] MrBUMP for cryo-EM

Hi Herman,

A path to a local copy of the PDB will work, although I should caution that 
MrBUMP's cryo-EM mode is releasing with ccp4 update 017 next week. This also 
corresponds with an update Ronan has made that allows MrBUMP to search the EBI 
Alphafold database, so that will also be available in the cryo-EM mode.

Best wishes,

Adam



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[ccp4bb] AW: [ccp4bb] COOT RSR

[Schreuder, Herman /DE]

Dear Paul,

I agree with Oliver and Norbert, in the early phases of refinement, when a lot 
of rebuilding has to be done, the coot RSR is not very helpful and in general I 
leave it to buster do refine the rebuilt regions. Knowing now that (dummy) 
waters may be the culprit, I will remove them. However, it would be much more 
convenient if the  repulsions with water molecules would not be taken into 
account.

Thank you,
Herman

Von: CCP4 bulletin board  Im Auftrag von Norbert Straeter
Gesendet: Sonntag, 21. November 2021 12:24
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] COOT RSR

Dear Paul,

I agree to Oliver. In situations where you want to fit a "dummy water" to a 
difference density peak (to determine distances to the environment) or try the 
fit of solvent molecules to yet unexplained density, it would be nice to first 
obtain an optimal fit and next think about the environment and possible bumps 
and partial occupancy. I had this problem already several times, that I could 
not get a good fit because of the model repulsions. Even if two partially 
occupied waters are at closed distance the repulsions appear to be active in 
RSR. It is not about the old method being better.

Best wishes,

Norbert



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[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] COOT RSR

[Schreuder, Herman /DE]

Hi Paul,

For me, dummy waters are waters placed in very early models, where it is not 
yet clear whether the density belongs to a water, a rearranged side chain or a 
ligand. In the course of the refinement process, it becomes clear to which 
category the density really belongs. Especially for larger rebuilding, I find 
it helpful to use such waters.

A real water is for me a nicely defined, hydrogen bonded water in a 
well-refined structure. However, in this stage one does not need any RSR 
anymore.

Best regards,
Herman

Von: CCP4 bulletin board  Im Auftrag von Paul Emsley
Gesendet: Montag, 22. November 2021 22:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] COOT RSR




What's the difference between a dummy water and a real one?

Paul


On 22/11/2021 14:34, Schreuder, Herman /DE wrote:
Dear Paul,

I agree with Oliver and Norbert, in the early phases of refinement, when a lot 
of rebuilding has to be done, the coot RSR is not very helpful and in general I 
leave it to buster do refine the rebuilt regions. Knowing now that (dummy) 
waters may be the culprit, I will remove them. However, it would be much more 
convenient if the  repulsions with water molecules would not be taken into 
account.

Thank you,
Herman

Von: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
Im Auftrag von Norbert Straeter
Gesendet: Sonntag, 21. November 2021 12:24
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] COOT RSR

Dear Paul,

I agree to Oliver. In situations where you want to fit a "dummy water" to a 
difference density peak (to determine distances to the environment) or try the 
fit of solvent molecules to yet unexplained density, it would be nice to first 
obtain an optimal fit and next think about the environment and possible bumps 
and partial occupancy. I had this problem already several times, that I could 
not get a good fit because of the model repulsions. Even if two partially 
occupied waters are at closed distance the repulsions appear to be active in 
RSR. It is not about the old method being better.

Best wishes,

Norbert



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[ccp4bb] AW: [ccp4bb] [EXTERNAL] Re: [ccp4bb] question using a model from a multiple model PDB file using Phaser

[Schreuder, Herman /DE]

Hi Yong,

Why make an ensemble when you don't want to search with and ensemble? You could 
run separate phaser jobs, one after the other, with your search models. I guess 
that this is what is meant by "SERIAL". Maybe Phaser has an option to do this, 
but it is also not difficult to write a small script to do this.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Yong Wang
Gesendet: Donnerstag, 9. Dezember 2021 22:28
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] question using a model from a 
multiple model PDB file using Phaser

Hi Randy,

I did not use the ensemble as I am not doing the typical jobs: I don't want an 
ensemble as a template, instead I would like to test each individual model 
separately. I re-formatted a multi-model pdb file (the original file did not 
have the MODEL line) on my own. Changing to "MODEL 0" did not change the error 
message. By the way, Phaser output suggested " SYNTAX ERROR: Use SERIAL". What 
is the usage of "SERIAL"? I can't find that keyword in the manual.

Thanks,

Yong

-Original Message-
From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Randy John Read
Sent: Thursday, December 9, 2021 4:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] question using a model from a multiple model 
PDB file using Phaser

EXTERNAL EMAIL: Use caution before replying, clicking links, and opening 
attachments.

Hi Yong,

If you make an ensemble with ensembler, the models are numbered from 1 to n, so 
there's no requirement to start from 0. Did you use ensembler to make your 
ensemble model file? If you did and Phaser is failing, could you send me the 
file so we can figure out the problem? Otherwise, the easiest solution might be 
to use ensembler to generate the ensemble!

Best wishes,

Randy Read

> On 9 Dec 2021, at 21:00, Yong Wang 
> <3c4fc05cc53b-dmarc-requ...@jiscmail.ac.uk>
>  wrote:
>
> Hi,
>
>
>
> The first model number in the pdb file is 1. In the script it is "MODEL 1". I 
> will try "MODEL 0" in the script to see how it goes.
>
>
>
> Thanks,
>
>
>
> Yong
>
>
>
> From: Boaz Shaanan mailto:bshaa...@bgu.ac.il>>
> Sent: Wednesday, December 8, 2021 7:37 AM
> To: Yong Wang mailto:wang_yon...@lilly.com>>
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: [EXTERNAL] Re: [ccp4bb] question using a model from a multiple model 
> PDB file using Phaser
>
>
>
> EXTERNAL EMAIL: Use caution before replying, clicking links, and opening 
> attachments.
>
>
> Hi,
>
> Did you try to edit 'Model 1' to 'Model 0'?
>
> I seem to recall that it was reported on this bb as the solution to this 
> problem.
>
> Cheers,
>
> Boaz
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben Gurion University
> Beer Sheva, Israel
>
>
>
> On 7 Dec 2021 22:56, Yong Wang 
> <3c4fc05cc53b-dmarc-requ...@jiscmail.ac.uk>
>  wrote:
>
> Hi All,
>
>
>
> I am interested in using individual models from a PDB file containing 
> multiple models with MODEL/ENDMDL records as a template for use in Phaser. 
> The usage in the manual "ENSEMBLE  [PDB|CIF]  [RMS 1 | 
> ID 2 | CARD ON3] CHAIN ""4 MODEL 5 " seems to support this. 
> However, I am getting an error shown below:
>
>
>
> ENSEmble mol PDB out.pdb RMS 1.5 MODEL 1
>
>
>
> $$
>
> 
>
> $TEXT:Warning: $$ Baubles Markup $$
>
> --
>
> SYNTAX ERROR: Use SERIAL
>
> --
>
> $$
>
>
>
> I am assuming that the error is from using the "MODEL 1" and not due to 
> something else. Does anyone have any suggestion on this problem?
>
>
>
> Thanks,
>
>
>
> Yong
>
>
>
>
>
>
>
> Yong Wang, Ph.D.
>
>
>
> Senior Research Advisor
>
> Global Structural Biology
>
> Lilly Research Laboratories
>
> Lilly Corporate Center
>
> Indianapolis, IN 46285
>
> U.S.A.
>
>
>
> Tel: +1 (317) 655 9145
>
>
>
>
>
>
>
>
>
> To unsubscribe from the CCP4BB list, click the following link:
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>

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building Fax: +44 1223 336827
Hills Road E-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Xray-dataset usable despite low completeness ?

[Schreuder, Herman /DE]

Dear Matthias,

I have the impression that you have set FRIEDEL'S_LAW= FALSE in your input, 
which means that CORRECT will print the anomalous completeness. If you plan to 
solve your structure with molrep, you can set  FRIEDEL'S_LAW= TRUE, which 
should give a much better completeness.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Matthias Oebbeke
Gesendet: Montag, 25. November 2019 14:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Xray-dataset usable despite low completeness ?

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear ccp4 Bulletin Board,

I collected a dataset at a synchrotron beamline and got the statistics
(CORRECT.LP) after processing (using xds) shown in the attached pdf-file.

Do you think this dataset is usable, due to its low completeness? As you can 
see in the attached file the completeness is just 50% in the highest resolution 
shell, whereas the I over Sigma is above 2 and also the CC 1/2 (80%) and the R 
factors (36.8%) have reasonable values.  
Furthermore the overall statistic are good regarding R factor, CC 1/2 and I 
over Sigma. The only problem seems to be the completeness. If I would set the 
cut-off at a lower resolution to get good completeness, I would throw away 
nearly half of my reflections.

I'm happy to here your opinion. In Addition to that: The space group is 
orthorhombic and the dataset was collected over 120° using fine slicing (0.1°).


Best regards,

Matthias Oebbeke


--
Matthias Oebbeke, M.Sc.
Research Group of Professor Dr. G. Klebe Institute of Pharmaceutical Chemistry 
Philipps-University Marburg Marbacher Weg 6, 35032 Marburg, Germany
Phone: +49-6421-28-21392
Mail: oebbe...@staff.uni-marburg.de
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.agklebe.de&d=DwIFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=idNV-Xu_XhSkWahyKK2ZqOTTVW-7hP4GKDQPejES6IM&s=5Ps7mkyXKGh0MHcdE62BdDx-glNcjfkXJ0hyAzzND-c&e=



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] 8/ 2263 spots indexed XDS

[Schreuder, Herman /DE]

Dear Almu,

were your data collected at a synchrotron? Many synchrotrons run automatic 
processing of the data being collected and it is worth looking if there is some 
processing directory between the frames you collected. If so, these data may 
already be quite decent, and in any case, the XDS.INP file may tell you how to 
set the optimal processing parameters for this particular beamline.

You can also look at the refined parameters of your successful XDS run, 
especially the ORGX and ORGY. Try to rerun XDS, using the refined parameters of 
the successful run.

Good luck!
Herman

Von: CCP4 bulletin board  Im Auftrag von Almudena Ponce 
Salvatierra
Gesendet: Freitag, 29. November 2019 12:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] 8/ 2263 spots indexed XDS


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear all,

I have some data sets that don't want to be processed :p

In one of them, when I look at IDXREF.LP I see virtually none of the found 
spots were indexed and the reason is that they are "too far from the expected 
position". The spots are smeary and elongated, so not the prettiest.

I have managed to process so far only one data set with decent statistics from 
another crystal harvested from the same drop, where the diffraction spots look 
better.

I am trying to find in the xds wiki the keyword I should fine tune in order to 
make those spots indexable.

Could you help me please?

Thank you very much in advance.

Best wishes,

Almu



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] 8/ 2263 spots indexed XDS

[Schreuder, Herman /DE]

Hi Almuda,
in addition to everything that had been said, I would have a look at all the 
images of your data set, to see if there is anything that may be causing 
misindexing in addition to the streaky spots. ADXV has the option to play a 
movie of a complete data set. Also, ORGX and ORGY must be accurate; as a crude 
estimate, the error should be less than half the distance between to 
diffraction spots. Also, where Clemens was hinting at, damaged detector pixels 
that are not removed from the image may jeopardize the indexing.
Best,
Herman

Von: Almudena Ponce Salvatierra 
Gesendet: Samstag, 30. November 2019 11:00
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [EXTERNAL] [ccp4bb] 8/ 2263 spots indexed XDS


EXTERNAL : Real sender is maps.fa...@gmail.com<mailto:maps.fa...@gmail.com>

Hi Herman,

thank you for your answer. Yes, my data were collected at the synchrotron, 
however, the autoprocessing failed with some of the datasets. I have checked 
the image headers and the XDS.INP files, trying to find any mismatches 
concerning origin, wavelength, etc... but it seems that the information was 
recorded properly. I am trying different origins with one of the datasets, but 
it has not helped so far.

I will write back once I find out what the problem was.

Best wishes,

Almudena

El vie., 29 nov. 2019 a las 14:07, Schreuder, Herman /DE 
(mailto:herman.schreu...@sanofi.com>>) escribió:
Dear Almu,

were your data collected at a synchrotron? Many synchrotrons run automatic 
processing of the data being collected and it is worth looking if there is some 
processing directory between the frames you collected. If so, these data may 
already be quite decent, and in any case, the XDS.INP file may tell you how to 
set the optimal processing parameters for this particular beamline.

You can also look at the refined parameters of your successful XDS run, 
especially the ORGX and ORGY. Try to rerun XDS, using the refined parameters of 
the successful run.

Good luck!
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Almudena Ponce Salvatierra
Gesendet: Freitag, 29. November 2019 12:48
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [EXTERNAL] [ccp4bb] 8/ 2263 spots indexed XDS


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

Dear all,

I have some data sets that don't want to be processed :p

In one of them, when I look at IDXREF.LP I see virtually none of the found 
spots were indexed and the reason is that they are "too far from the expected 
position". The spots are smeary and elongated, so not the prettiest.

I have managed to process so far only one data set with decent statistics from 
another crystal harvested from the same drop, where the diffraction spots look 
better.

I am trying to find in the xds wiki the keyword I should fine tune in order to 
make those spots indexable.

Could you help me please?

Thank you very much in advance.

Best wishes,

Almu



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Problem with structural alignment

[Schreuder, Herman /DE]

Dear Ishan,

A structural alignment should be independent of the software used.
However, the residues being selected for the alignment can make a big 
difference and different programs have different selection criteria.
There are two cases:

  1.  Different proteins. Here one needs to decide what the equivalent residues 
are. Usually this is based on a sequence alignment, but once a crude 
superposition has been obtained, the equivalent residue pairs can be optimized.
  2.  The same protein undergoing a conformation change (e.g. loop or domain 
movement). Here one has to decide what the rigid regions are and where the 
moving loop or linker is. By looking at the coarse superposition with coot or 
any other graphics program, one quickly sees what would be the best borders.

If it is really critical for your hypothesis, you will have to define manually 
the equivalent pairs.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Eleanor Dodson
Gesendet: Montag, 16. Dezember 2019 11:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Problem with structural alignment


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Well CCP4mg does this very nicely and it is easy to check the results - all 
displayed on the screen so you can pinpoint outliers..
Eleanor

On Mon, 16 Dec 2019 at 06:38, Ishan Rathore 
mailto:ishanrathor...@gmail.com>> wrote:
Hi,

I am trying to compare multiple homologous structures of a protein, where I am 
analysing the active site residues and the bound substrate/peptide. I have used 
multiple methods for alignment in coot and pymol. Every method gives a slightly 
different orientation in the active site. Based on the analysis I am trying to 
propose a hypothesis for the catalytic mechanism of the protein. But, I am a 
bit wary of getting biased with the alignment if that supports my hypothesis.

What are the parameters that have to be considered for a reliable alignment?
What are the other Softwares available for alignment?



Thanks and regards
Ishan



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Potential weak binding ligand in the active site

[Schreuder, Herman /DE]

Hi Katrin,

First, I think you meant that the green density disappears after contouring at 
6 Sigma and not 6 A?
That you ligand is only partly visible due to disorder and has partial 
occupancy happens often and is no reason for concern. Of course, you could try 
soaking with a 10-fold higher ligand concentration of that would be possible.
The approach you use: fit would you see and don’t fit what you don’t see is 
also correct. However, as you come closer to the noise level of your electron 
density map, you have to be more careful not to introduce model bias and 
artefacts. What I would to copy your current pdb file to another pdb file, 
delete the ligand you fitted and any water molecules near the ligand and run a 
few rounds of refinement to get an unbiased omit map. Then view this map 
together with your refined ligand a see if it makes sense.

Good luck and happy holidays!
Herman

Von: CCP4 bulletin board  Im Auftrag von Katherine Lim
Gesendet: Freitag, 20. Dezember 2019 05:57
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Potential weak binding ligand in the active site


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Hi all,

I apologise in advance for the long post. I am working on solving a structure 
that looks like it could have a ligand bound in the active site. My data was 
obtained from a crystal of just the soluble domain of my protein that had been 
soaked overnight in the ligand solution. The apo crystal structure is already 
known and so I have solved my structure using phaser MR. I have attached the 
Aimless report output of my structure at the end of this email. The current R 
values I have after refinement and adding in all the waters are Rfree: 0.2413 
and Rwork: 0.1897. I can clearly see green density that is much larger (only 
disappears when I contour the Fo-Fc map to about 6 A) than in my control 
crystal that had been soaked in the same concentration of just solvent (I had 
used DMF).

I am struggling to add in my ligand as it doesn't seem like the entire ligand 
can fit in the green density. We think it may be because we have only used the 
soluble domain and so the ligand isn't held very securely since the 
transmembrane domain is missing. I have been trying to fit in smaller sections 
of it and doing an occupancy refinement. So far I have been able to get part of 
the ligand in with an occupancy of about 0.6 but after the refinement run, 
phenix.refine seems to move this part of the ligand slightly out of the area 
where I had tried to fit it into the green density. Interestingly, there isn't 
a big red density in the area that this ligand section has moved to. I would 
appreciate any advice on how I should proceed with trying to figure out if I 
have tried the correct section of the ligand and the kind of refinement 
settings to use with a weak binder.

Space Group
P212121
Unit cell abc
84.76, 89.84, 91.55
unit cell alpha beta gamma
90, 90, 90
OVERALL
LOW RES
HIGH RES
Low res limit
45.78
45.78
1.94
High res limit
1.9
9.11
1.9
Rmerge
0.234
0.052
1.684
Rmeas
0.244
0.054
1.768
Rpim
0.069
0.016
0.527
Total # observations
663073
6282
36851
Total # unique
55174
579
3359
I/sigma
7.5
27.9
1.4
CC ½
0.997
0.999
0.747
Completeness %
99
98.7
94.7
Multiplicity
12
10.8
11

Katherine Lim

PhD Candidate

School of Biomedical Sciences; School of Molecular Sciences

Marshall Centre for Infectious Disease Research and Training

The University of Western Australia



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[ccp4bb] AW: Mixed oligomeric states in crystallo

[Schreuder, Herman /DE]

Dear Thomas,

The asymmetric unit of the bovine pTAFI structure (3dgv) contains one regular 
dimer and one somewhat disordered monomer. However there is no domain-swapping 
of alpha-helices or other special things.

Best regards,
Herman


Von: CCP4 bulletin board  Im Auftrag von Kluenemann, 
Thomas
Gesendet: Freitag, 31. Januar 2020 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Mixed oligomeric states in crystallo


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear all,

We recently solved a the structure of a small c-type cytochrome. We observed, 
that of the eleven chains in the asymmetric unit ten form 3D domain swapped 
dimers by exchanging an α-helix. The eleventh  chain is present as a monomer. 
Based on the anomalous iron signal and the chain tracing we are sure that no 
chain was missed.
I tried to find other examples in the PDB, were one crystal is made of 
different homo- or heterooligomers.  I only found proteins with partial 
occupied peptide binding sites, which is not what I am looking for. Does anyone 
know of a case were the presence of different homo- or heterooligomers is 
required to form the crystal?

Best regards,
Thomas Klünemann





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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Macromolecular Crystallography workshop in South America 2020

[Schreuder, Herman /DE]

Dear Bärbel,

this is a good point. Last fall, I attended the bachelors ceremony for 
informatics students for my daughter. I was somewhat surprised to see that most 
German graduates were male, but that most Indian graduates were female. At the 
reception afterwards when talking to some people I discovered that in Germany, 
an informatics study is considered to be something for males, while in India it 
is apparently considered to be something for females. In fact, some of the 
Indian students where somewhat surprised that so little German females would 
study informatics.

So apparently, a lot of the gender bias is the result of cultural expectations 
and this will be the first thing we need to change if we want to get a more 
equal gender distribution.

Best regards,
Herman


Von: CCP4 bulletin board  Im Auftrag von Bärbel Blaum
Gesendet: Donnerstag, 6. Februar 2020 10:31
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Macromolecular Crystallography workshop in 
South America 2020


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear all,

yes, there are more male computational crystallographers than females – the 
question we can ask ourselves here is: Do we think there is some biological 
reason for this? Obviously there isn’t. So things are wrong as they stand. Are 
we, nevertheless, ok with this current state of affairs? We are not talking 
some minor issue here – if half of the population is under-represented, that is 
a giant intellectual loss (actually for *all* professions that lack diversity, 
childcare included, and of course the loss is not just on the intellectual 
level).

If you think you have been lucky to work in a place where there is no bias 
against women it just means you are lucky in not being one. If you care to 
know, simply try and put the other side’s shoes on. Start asking your female 
colleagues, friends, partners, students about their experience, moments they 
witnessed were they felt overlooked or deliberately excluded, treated in sexist 
ways, you name it. You will be surprised.

Then think about the moments, institutions, relationships that have been 
instrumental in your own careers. University meetings and important talks 
routinely held and given in the evenings. Conferences with no childcare. Many 
male researchers only being able to work as much as they do because some female 
is looking after the house and the kids. Babies being colored-coded by gender, 
mummys at home and daddys at work. Girls being told it’s ok to be bad at math. 
You must be joking if you wonder where all the female scientists are! We all 
grow up and are educated in a world that is heavily biased in terms of which 
roles which gender adopts - how are we supposed to not think that this reflects 
some form of “natural” order? There are plenty of other groups that grow up 
systematically underestimating their potentials, just compare students from 
academic homes with those from working class backgrounds. And there is plenty 
of research on the subject.

The question is if you care to notice this bias, discrimination, lack of equal 
opportunities, and if you care to do something about it - of course we all have 
other things to attend to. But if teaching is part of our job description then 
we have to think about how to be good teachers, and that does not stop at how 
to explain Fourier transforms or python. The reason I suggested a meeting by a 
female group of scientists as only organizers and speakers is because as long 
as we have these male-dominated environments at workshops and meetings the 
respective younger generation will be presented with this order as being the 
natural way things work *because we taught them so*. If we want these younger 
guys to get things right in ways we did not we have to create alternative 
perspectives and come up with new forms of community work.

Bärbel

--
Bärbel Blaum, PhD
Inthera Bioscience AG
Einsiedlerstrasse 34
CH-8820 Waedenswil
Switzerland
E-Mail: baerbel.bl...@intherabio.com
Phone: +41 43 477 94 72--



Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
im Auftrag von Susan Lea 
mailto:susan@path.ox.ac.uk>>
Antworten an: Susan Lea 
mailto:susan@path.ox.ac.uk>>
Datum: Donnerstag, 6. Februar 2020 um 02:21
An: mailto:CCP4BB@JISCMAIL.AC.UK>>
Betreff: Re: [ccp4bb] Macromolecular Crystallography workshop in South America 
2020

Perhaps some of the respondents to this question should real Angela Saini’s 
Inferior before commenting in public
Susan
Sent from my iPhone

On 5 Feb 2020, at 19:18, Robert Nicholls 
mailto:nicho...@mrc-lmb.cam.ac.uk>> wrote:

As a Caucasian male I hesitate to post; I know that there are a lot of people 
who are sensitive to this subject, so feel that I'm treading on eggshells in 
responding... Nevertheless I feel obliged to respond, having a certain amount 
of insight into the topic in the context of these workshops.

Personall

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Lattice-translocation defect (LTD)

[Schreuder, Herman /DE]

Hi Daniele,

I agree with Wim that the first thing you should check is your space group and 
especially whether a ncs symmetry element has been mistakenly identified as 
being crystallographic. Since your Patterson peak is along w (c-axis), you have 
to change the space group for processing such, that there are no rotation axes 
or other symmetry elements perpendicular to the c-axis any more. If you have a 
low-symmetry space group, you could also process in P1 to be absolutely on the 
safe side. Than you should run MR in this lower symmetry space group.

Concerning lattice translocation defects, almost every case is unique and I am 
not aware of any software able to handle this. Here you will have to work out 
the maths for your particular case yourself and apply the correction with e.g. 
sftools. I am a little puzzled by your 7Å vector in your (native?) Patterson 
maps. I guess, if you translate your protein by 7Å it will strongly overlap 
with itself and I guess the same will be true for your ghost map. You should 
also have a look at your diffraction images, perhaps after summing them to 1° 
slices so become human-interpretable. In many cases, LTD is associated with a 
mix of sharp and fuzzy diffraction spots. Seeing those would be a strong 
indicator that you have the problem, but there are cases of LTD where all the 
spots are sharp. You may also want to check for statistical disorder.

Good luck!
Herman

Von: CCP4 bulletin board  Im Auftrag von Wim Burmeister
Gesendet: Mittwoch, 12. Februar 2020 08:57
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Lattice-translocation defect (LTD)


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk


Hello,

do you have some details about the space group ? Did the integration not miss 
any sports ? I would rather think of an ncs close to crystallographic symmetry, 
or maybe some twinning problem.

I guess these are Pilatus data, can you combine the frames into 1 degree 
oscillations and try Mosflm processing to see how the patterns integrate ?

Greetings

Wim
Le 11/02/2020 à 22:31, Daniele de Sanctis a écrit :
Hi all,

I'm currently dealing with what I think it is a case of LTD (off-origin 
Patterson peak, with vector along w of ~ 7A and electron density map showing a 
"ghost" map shifted by 7 A). I saw there are quite a few cases reported in 
literature  (for example Hare et al, 2006), but what I could not find is how I 
can demodulate the data. Is there any software that can be used for this?

Thank you

Daniele

--
ἀρετή
---
Daniele de Sanctis, PhD

Structural Biology Group
ESRF, Grenoble, France
Tel 33 (0)4 76 88 2869



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--
Wim Burmeister

Professeur
Institut de Biologie Structurale (IBS) CIBB
71 avenue des Martyrs / CS 20192
38044 Grenoble Cedex 9, FRANCE
E-mail: wim.burmeis...@ibs.fr
Tel:+33 (0) 457 42 87 41   Fax: +33 (0) 476 20 94 00
website

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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] problem in structure solution of multidomain protein

[Schreuder, Herman /DE]

Dear Rajnesh,

My experience with molecular replacement is that when you don’t have a model, 
you don’t get density. Only in exceptional cases (crystals with a very high 
solvent content) I see density for missing loops or domains, so missing density 
is very inconvenient, but no reason to be worried.

You say that with the 4 molecule search model you get a MR solution and 
density, but no density for the 5th molecule.
With the model for the 5th molecule, you also get a MR solution, but no density 
for molecules 1-4.

What happens if you load in coot both the solution for the 4 molecules and the 
solution for the 5th molecule? Does that produce a sensible crystal packing 
(e.g. no serious overlaps)? The solutions may be in different asymmetric units, 
so you may have to apply some crystallographic symmetry transformations to 
bring them together. If the crystal packing makes sense, you could merge the 
pdb files and see if you can refine the combined solutions.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Eleanor Dodson
Gesendet: Mittwoch, 12. Februar 2020 14:04
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] problem in structure solution of multidomain 
protein


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

At 3A  finding missing domains is tricky.. Can you increase that resolution at 
all? Much easier at 2.5A!
But I would refine and rebuild the 4 domains   to the best possible maps, then 
see if there is any density unaccounted for.
(You will have to lower the COOT default contour level I guess..)

If there is you could use buccaneer to try and build it, keeping the fitted 
domains fixed ..
That can work IF there is density to build into..
Presumably you know the sequence?
MR from a modelling model is often tricky anyway.
Eleanor


On Wed, 12 Feb 2020 at 12:42, Boaz Shaanan 
mailto:bshaa...@bgu.ac.il>> wrote:
Hi,
One other option is to run MR in Phaser in one run with the tetramer made of 
the 4 monomers as model 1 and the single monomer as model 2. The gui offers 
this option and there is also an example in the documentaion.
Cheers,
Boaz
Boaz Shaanan, Ph.D.
Department of Life Sciences
Ben Gurion University of the Negev
Beer Sheva
Israel

On Feb 12, 2020 09:23, Rajnesh Kumari Yadav 
mailto:rajn...@rcb.res.in>> wrote:
Hello everyone,

I am working on a protein which have 5 domain in it, we have its 4 domain 
structure and 3.0 Angstrom data of 5 domain protein crystal. While doing the 
Molecular replacement we got solution with four domain with good electron 
density and space for last domain without any density. When we tried Molecular 
replacement with the missing domain (modelled) only we got solution shows 
density for this missing domain but not able see electron density for rest four 
domain even though room for 4 domain was visible in the solution. I need help 
or suggestion for this issue.



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[ccp4bb] AW: [ccp4bb] What resolution - X-ray diffraction round this time

[Schreuder, Herman /DE]

I fully agree with this!
Best, Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Phoebe A. Rice
Gesendet: Freitag, 28. Februar 2020 17:03
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] What resolution - X-ray diffraction round this 
time

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Can we get some momentum for the "standard table 1" including TWO numbers - 
outer limit used in refinement, and nominal resolution based on some standard 
such as I/sigI =2 (or 3, or whatever the community can agree on)?  That would 
hopefully cut down on all the reviewer complaints of overstated resolution.



~~~

Phoebe A. Rice

Dept. of Biochem & Mol. Biol. and

  Committee on Microbiology

https://urldefense.proofpoint.com/v2/url?u=https-3A__voices.uchicago.edu_phoebericelab_&d=DwIGaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=lVmKxr1h6ivkmcYp1sQ_nzmWIfbFS5XWaQMHhaxbeeM&s=CXNbP2DMixZriXPv1R2bTMMTnMPdy4mwce_8MJuNxlA&e=

 



On 2/28/20, 6:56 AM, "CCP4 bulletin board on behalf of Malý Martin" 
 wrote:



Dear colleagues,



I agree with all the previous responses, it is a pity to throw away

useful high-resolution data. The problem of high-resolution cutoff

estimation is also nicely summarized in another paper by Andrew Karplus

and Kay Diederichs "Assessing and maximizing data quality in

macromolecular crystallography"


https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih.gov_pmc_articles_PMC4684713_&d=DwIGaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=lVmKxr1h6ivkmcYp1sQ_nzmWIfbFS5XWaQMHhaxbeeM&s=lrf35lEnkEOs6pHot-cjxf9f3SI4dmCvkcL4098veOQ&e=
 . It is suggested

using CC1/2 for the selection of the cutoff for data processing (not

I/sigI or R_whatever). Later on, the decision should be validated

performing the paired refinement protocol.



Good luck with the argumentation.

Martin





On 2/28/20 11:08 AM, LMB wrote:

> Ask the referee - (apart from the other suggestions here)

>

> ‘How would removing data Improve my model?”

>

> Sent from my iPad

>

>> On 28 Feb 2020, at 08:22, dusan turk  wrote:

>>

>> Hi,

>>

>> Browsing through the recent discussion on EM data resolution cutoff

>> it occurred to me that the X-ray diffraction community isn’t that

>> unanimous either.

>>

>> My stand:

>>

>> When the default resolution cutoff provided with the data processing

>> software in electron density map calculation and refinement delivers

>> quality maps noisier than expected and/or too high R-factors I start

>> adjusting the resolution cutoff by lowering the resolution and trying

>> alternative space group. Hence, I allow the data processing programs

>> to suggest where to draw the line (be it CC1/2, I/sigI, R merge, R

>> sym, R p.i.m. and R r.i.m, …) , unless there are problems.

>>

>> Doing so, I came into a dispute with a referee who shaped his request:

>>

>> "It is well accepted that the criteria for resolution cutoff should

>> consider both I/SigI and Rmerge for the outer most shell. For data

>> sets collected at synchrotron sources, the criteria of I/SigI > 5 and

>> Rmerge <50% can be taken as a good practical reference.”

>>

>> So where do we stand? Which are the most objective criteria for

>> resolution cutoff to be used in diffraction data processing? Which

>> number of shells to use when calculating the statistics? Do we have a

>> consensus?

>>

>> best wishes,

>>

>> dusan turk

>>

>>

>>

>> Dr. Dusan Turk, Prof.

>> Head of Structural Biology Group 
https://urldefense.proofpoint.com/v2/url?u=http-3A__stef.ijs.si_&d=DwIGaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=lVmKxr1h6ivkmcYp1sQ_nzmWIfbFS5XWaQMHhaxbeeM&s=_3e4jARMhVBSasLY6-m9dOnl_vTRnBGwLgzq_9Dv6c8&e=

>> Head of Centre for Protein and Structure Production

>> Centre of excellence for Integrated Approaches in Chemistry and

>> Biology of Proteins, Scientific Director

>> 
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.cipkebip.org_&d=DwIGaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=lVmKxr1h6ivkmcYp1sQ_nzmWIfbFS5XWaQMHhaxbeeM&s=78xv1Oze-kc7Gv9avvfnElFBluCHaasC65gzTDktaDM&e=

>> e-mail: dusan.t...@ijs.si

>> phone: +386 1 477 3857 Dept. of Biochem.& Mol.& Struct. Biology

>> fax: +386 1 477 3984 Jozef Stefan Institute

>> Jamova 39, 1 000 Ljubljana,Slovenia

>> Skype: dusan.turk (voice over internet: 
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.skype.com&d=DwIGaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdyw

[ccp4bb] AW: [EXTERNAL] [ccp4bb] An error in the IUCr Online Dictionary of Crystallography

[Schreuder, Herman /DE]

Dear Gerhard,
By clicking on the "Actions" button, I discovered that the contribution comes 
from Brian McMahon. Maybe you could contact him about this?
Best regards,
Herman 


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Gerard Bricogne
Gesendet: Dienstag, 10. März 2020 15:16
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] An error in the IUCr Online Dictionary of 
Crystallography

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear all,

 While we are all discussing how to "push the frontiers" of our field, 
there seem to be some dark and dusty corners in the documentation of some basic 
notions. For instance the formula for the real-space correlation coefficient 
given at 

 
https://urldefense.proofpoint.com/v2/url?u=https-3A__dictionary.iucr.org_Real-2Dspace-5Fcorrelation-5Fcoefficient&d=DwIBAg&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=1AJ1T0r6mgUdK4idVKIjexWpgp_ApLYGh_-nN1mePPo&s=MizVfSbJiL7RRu5wYQy9khDyX9qrR2dEs_vzAIdQiHk&e=

is completely garbled. The absolute value signs in the denominator are 
superfluous, although harmless since we are dealing with real numbers, but the 
numerator is wrong in two respects:

 1. there should not be absolute values around the deviations from the
mean of the two types of densities;

 2. the expression should be a single summation of products of those two
types of deviation, not a product of single sums!

Does anyone know how to get this corrected?


 With best wishes,

  Gerard.



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus

[Schreuder, Herman /DE]

Dear Chitra,

There usually is a reason the C-terminus is disordered. Either it needs to bind 
a ligand to get ordered, or it needs to bind to some other protein. You have to 
check the literature. If the C-terminus binds a ligand, you have to add this 
ligand to your crystallization experiment. If it binds to some other protein, 
you could try cocrystallization of both proteins, or just try to cocrystallize 
the other protein with only the 20 residues or so of the C-terminus.

Best, Herman

Von: CCP4 bulletin board  Im Auftrag von chitra latka
Gesendet: Donnerstag, 12. März 2020 08:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus

[Schreuder, Herman /DE]

Dear Chitra,

Crystallizing both proteins together is a very good idea: If you get a 
structure, it will be an interesting one, even if the C-terminus remains 
invisible.

Concerning the flexible C-terminus: is it the linker linking the second domain 
to the first one? In that case it might just be a bait, attracting a protease 
to get the second domain chopped off. In that case, chances of getting it 
structured may be slim and if you get it structured, it might be a crystal 
packing artifact. However, as Klemens mentioned, you should nevertheless try!

If this flexible C-terminus gets cleaved by a protease, you may also try to get 
some cocrystal structure with the protease. There are quite a few tricks 
available to achieve this.

Best, Herman



Von: chitra latka 
Gesendet: Donnerstag, 12. März 2020 12:59
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is chitra.la...@gmail.com<mailto:chitra.la...@gmail.com>

Dear Herman,

Its a two domain protein and the second domain gets chopped off and stabilises 
the other domain by binding near the flexible C - terminus of first domain. I 
am trying to crystallise both the proteins together. Literature review doesn't 
report binding of the protein to any other protein. So, it kind of has the 
ligand or another domain to stabilise but eve then density of C-terminus 
residues remain missing.

Thanks
Chitra Latka










On Thu, Mar 12, 2020 at 4:08 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Chitra,

There usually is a reason the C-terminus is disordered. Either it needs to bind 
a ligand to get ordered, or it needs to bind to some other protein. You have to 
check the literature. If the C-terminus binds a ligand, you have to add this 
ligand to your crystallization experiment. If it binds to some other protein, 
you could try cocrystallization of both proteins, or just try to cocrystallize 
the other protein with only the 20 residues or so of the C-terminus.

Best, Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von chitra latka
Gesendet: Donnerstag, 12. März 2020 08:54
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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--
Regards
Chitra



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[ccp4bb] Wincoot middle-mouse button does not work

[Schreuder, Herman /DE]

Dear community,

Courtesy to the Corona crisis, I am now separated from my Linux workstation and 
I am trying to get everything working from home. Running coot remotely via a 
low-speed internet connection was no option so I had Wincoot installed on my 
laptop. Everything appears to work fine with one exception: The middle-mouse 
button is not working, so I cannot center on an atom.

Does anyone know how to solve this problem?

Thank you for your help and stay healthy!
Herman






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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work

[Schreuder, Herman /DE]

Hi Arne (and others),

I am using Windows 10 und just downloaded WinCoot 0.8.9.2 EL. Since everybody 
else’s mouses seem to be perfectly fine, I will try another mouse. Maybe the 
one I am using is having a problem.

Best,
Herman

Von: EchelonIV . 
Gesendet: Montag, 23. März 2020 16:34
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work


EXTERNAL : Real sender is 
arne.raasa...@gmail.com<mailto:arne.raasa...@gmail.com>

Hi Herman,

which version, OS and mouse are you using? I can confirm that middle mouse 
button works perfectly fine in version 0.8.6.2 (yes I know I'm outdated) win 
Win10 with a generic Logitech plug-and-play mouse with the third mouse button 
being the wheel. I also quickly checked the latest version in Windows 7 
ultimate (yikes!) with the same generic mouse as well as a fancy Razer mouse 
with cloud-based button profiles and it seems to work equally well. Have you 
checked your mouse settings and drivers?

Stay healthy,
Arne

On Mon, Mar 23, 2020 at 3:48 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear community,

Courtesy to the Corona crisis, I am now separated from my Linux workstation and 
I am trying to get everything working from home. Running coot remotely via a 
low-speed internet connection was no option so I had Wincoot installed on my 
laptop. Everything appears to work fine with one exception: The middle-mouse 
button is not working, so I cannot center on an atom.

Does anyone know how to solve this problem?

Thank you for your help and stay healthy!
Herman






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--
--
Arne Raasakka
PhD Biochemistry
Department of Biomedicine, University of Bergen
Norway



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[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work

[Schreuder, Herman /DE]

Dear all,

I just tried another mouse and now everything works as it should. Probably the 
mouse I was using before was configured differently but anyways, the problem is 
solved.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Roger Rowlett
Gesendet: Montag, 23. März 2020 17:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button 
does not work


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

For mice with a pressable scroll wheel it should be possible to go into the 
configuration settings to enable the scroll wheel press to be a middle click. 
It is typically not set to that function by default.

For two button mice, it is usually possible to configure a L-R press to emulate 
middle-click. Again, you have to go into the mouse configuration settings.

I have two mice (a 3-button and a 2-button) configured to work properly with 
Wincoot in Win10.


Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Colgate University

On Mon, Mar 23, 2020, 11:56 AM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Hi Arne (and others),

I am using Windows 10 und just downloaded WinCoot 0.8.9.2 EL. Since everybody 
else’s mouses seem to be perfectly fine, I will try another mouse. Maybe the 
one I am using is having a problem.

Best,
Herman

Von: EchelonIV . mailto:arne.raasa...@gmail.com>>
Gesendet: Montag, 23. März 2020 16:34
An: Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>>
Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
Betreff: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work


EXTERNAL : Real sender is 
arne.raasa...@gmail.com<mailto:arne.raasa...@gmail.com>

Hi Herman,

which version, OS and mouse are you using? I can confirm that middle mouse 
button works perfectly fine in version 0.8.6.2 (yes I know I'm outdated) win 
Win10 with a generic Logitech plug-and-play mouse with the third mouse button 
being the wheel. I also quickly checked the latest version in Windows 7 
ultimate (yikes!) with the same generic mouse as well as a fancy Razer mouse 
with cloud-based button profiles and it seems to work equally well. Have you 
checked your mouse settings and drivers?

Stay healthy,
Arne

On Mon, Mar 23, 2020 at 3:48 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear community,

Courtesy to the Corona crisis, I am now separated from my Linux workstation and 
I am trying to get everything working from home. Running coot remotely via a 
low-speed internet connection was no option so I had Wincoot installed on my 
laptop. Everything appears to work fine with one exception: The middle-mouse 
button is not working, so I cannot center on an atom.

Does anyone know how to solve this problem?

Thank you for your help and stay healthy!
Herman






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--
--
Arne Raasakka
PhD Biochemistry
Department of Biomedicine, University of Bergen
Norway



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!

[Schreuder, Herman /DE]

Dear Frank,

here I disagree. I think it is bad practice to complain to the editors or start 
naming and shaming before asking the authors first. Only if they do not want to 
cooperate, it would be time to bring the flame-throwers in position.

However, I think the situation is more subtle and that that is the reason Artem 
wrote his email: He wants the data, but does not want to reveal his identity to 
his competitors, who apparently made a significant effort not to reveal any 
useful details.

Here I would let me guide by the (perceived) commercial interest: if it is not 
gigantic, then I would contact the authors to prevent a wasteful duplication of 
research efforts.
If there is a significant commercial interest, it would probably be better not 
to contact them and go the hard way of solving the structure yourself. A thing 
to consider is also what would happen if the authors still would refuse: they 
know the identity of the competitor and you still do not have the data.

An alternative may be to ask an academic friend who also works on sausage 
esterases to inquire with the authors…

Good luck with your decision!
Herman

Von: CCP4 bulletin board  Im Auftrag von Frank von Delft
Gesendet: Dienstag, 7. April 2020 17:19
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Write to the editor - that's their job.

Though they may also see it as their job to ignore emails like yours because 
that's far easier than dealing with them.

Alternatively, go the Rupp Route:  Name and Shame! ;)

On 07/04/2020 16:08, Artem Evdokimov wrote:
Dear CCP4ers,

I would like to solicit your thoughts on the following (this is a real 
situation, but salient details are changed):

Imagine that you're an industrial scientist in a small company, working on the 
Bavarian Sausage (Weisswurst) Esterase project. The overall structure is 
previously unknown, with no good homologs in the PDB, trying to model is "OK 
not great" so the structure is really needed...

Then, you find an article from a large commercial competitor, that somehow 
managed to solve the Stadtwurst (Saxony Sausage) Esterase structure (which is a 
very close homolog to the one you need!).

Sounds good - but as you read the paper you realize that the authors managed to 
find a journal that allowed them to publish their work without disclosing 
neither the coordinates of the model, nor even the crystallization conditions 
of the protein - all that's available is a tantalizing still picture of the 
active site in surface mode, with a ball-and-stick ligand positioned such that 
it is impossible to say what it interacts with.

So you sit and ponder - whether to write to the Editor, or maybe to contact the 
authors directly (but then they would know that you're working on this, which 
is not necessarily great since you're competing), or to just buck up and do the 
structure on your own (which feels a bit wasteful). Then, you realize that your 
friends at CCP4 have a lot of wisdom to offer, so you sit down and pen an 
email...

Any thoughts?

Artem



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[ccp4bb] AW: AW: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!

[Schreuder, Herman /DE]

In this way I fully agree. To me it sounds like one of those journals that will 
publish anything without going through the trouble of peer review. However, 
this will not get Artem the coordinates he was looking for.

Best Herman

Von: Frank von Delft 
Gesendet: Dienstag, 7. April 2020 19:10
An: Schreuder, Herman /DE ; CCP4BB@JISCMAIL.AC.UK
Betreff: Re: AW: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!


EXTERNAL : Real sender is 
frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>

I meant:  complain to the editor for accepting a paper without released 
coordinates.  We as a community fought and won that battle >20 years ago - so 
we have the weight of history on our side.

On 07/04/2020 17:05, Schreuder, Herman /DE wrote:
Dear Frank,

here I disagree. I think it is bad practice to complain to the editors or start 
naming and shaming before asking the authors first. Only if they do not want to 
cooperate, it would be time to bring the flame-throwers in position.

However, I think the situation is more subtle and that that is the reason Artem 
wrote his email: He wants the data, but does not want to reveal his identity to 
his competitors, who apparently made a significant effort not to reveal any 
useful details.

Here I would let me guide by the (perceived) commercial interest: if it is not 
gigantic, then I would contact the authors to prevent a wasteful duplication of 
research efforts.
If there is a significant commercial interest, it would probably be better not 
to contact them and go the hard way of solving the structure yourself. A thing 
to consider is also what would happen if the authors still would refuse: they 
know the identity of the competitor and you still do not have the data.

An alternative may be to ask an academic friend who also works on sausage 
esterases to inquire with the authors…

Good luck with your decision!
Herman

Von: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
Im Auftrag von Frank von Delft
Gesendet: Dienstag, 7. April 2020 17:19
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk>

Write to the editor - that's their job.

Though they may also see it as their job to ignore emails like yours because 
that's far easier than dealing with them.

Alternatively, go the Rupp Route:  Name and Shame! ;)


On 07/04/2020 16:08, Artem Evdokimov wrote:
Dear CCP4ers,

I would like to solicit your thoughts on the following (this is a real 
situation, but salient details are changed):

Imagine that you're an industrial scientist in a small company, working on the 
Bavarian Sausage (Weisswurst) Esterase project. The overall structure is 
previously unknown, with no good homologs in the PDB, trying to model is "OK 
not great" so the structure is really needed...

Then, you find an article from a large commercial competitor, that somehow 
managed to solve the Stadtwurst (Saxony Sausage) Esterase structure (which is a 
very close homolog to the one you need!).

Sounds good - but as you read the paper you realize that the authors managed to 
find a journal that allowed them to publish their work without disclosing 
neither the coordinates of the model, nor even the crystallization conditions 
of the protein - all that's available is a tantalizing still picture of the 
active site in surface mode, with a ball-and-stick ligand positioned such that 
it is impossible to say what it interacts with.

So you sit and ponder - whether to write to the Editor, or maybe to contact the 
authors directly (but then they would know that you're working on this, which 
is not necessarily great since you're competing), or to just buck up and do the 
structure on your own (which feels a bit wasteful). Then, you realize that your 
friends at CCP4 have a lot of wisdom to offer, so you sit down and pen an 
email...

Any thoughts?

Artem



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Average B factors with TLS

[Schreuder, Herman /DE]

This is a very good point. I never was very happy with calculating the average 
of all B-factors. E.g. if one adds a lot of high-B water molecules, the average 
B goes up, but with the structure itself nothing changes. Instead of 
calculating the average B, it would probably better to calculate the “Wilson 
B-factor” of the calculated intensities and compare this to the observed Wilson 
B-factor.

My 2 cts, Herman

Von: CCP4 bulletin board  Im Auftrag von Edward Berry
Gesendet: Mittwoch, 8. April 2020 03:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Average B factors with TLS

EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Apologies for my previous email appearing to put words in Dale's mouth- I'm 
using my school's
webmail and it apparently doesn't indicate the quoted text.
The following is what I added:

I think it is not just that the distribution is asymmetric and limited to 
positive numbers-
it is due to the fact that "log" and "average" do not commute (the average of 
the logs
is not the log of the averages), and the logarithmic/exponential relation 
between intensity and B.

The wilson B is obtained from the slope of ln vs S^2.
from the relation  ~ exp(-2BS^2).
We can take that slope as the rise over run in the range from S=0 to s=1/(3A), 
even though
the real Wilson plot will follow it only around 3A and beyond.

 in a shell at resolution S will be down by a factor of
 exp(-2BS^2)  compared to  at S=0
for S=(1/3A) this is a factor of exp(-0.222*B)
   for B=10, this is a factor of 0.108
   for B=100, this will be a factor of 2.3E-10

Now if for half the atoms B is 10 and for the other half B is 100
 at 3 A will be something like (.108 + 2.3E-10)/2 = 0.054
(This is a little over my head because we are combining contribution of the
two sets of atoms to each reflection in the shell, and they add vectorially.
Maybe a factor of sqrt(2) is appropriate for random phases.
But for order of magnitude:)

The slope from S^2=0 to S^2=(1/3A)^2 =
-2B ={ ln(0.054) - ln(1) }/{. - 0}
-2B =  -26.3
B(wilson) = 13.2
Bave = (10+100)/2 = 55
The log of the average is larger than the average of the logs.

Another way of looking at it, the contribution of those atoms with B=100 at 3A 
is completely negligible compared to the contribution of the atoms
with B=10, so the slope around 3A reflects only the B-factor of the 
well-ordered atoms, and the slope measured there should give B=10, not 13.

If atoms were randomly distributed and we could apply Wilson over the  entire 
resol range, we still wouldn't get a straight line if there is a range of 
atomic B-factors.
It would be like "curve peeling" in analyzing two simultaneous first-order 
reactions with different half-time: semilog plot of dissociation of a mixture 
of fast and slow hemoglobin. Near zero time the curve is steep as the fast 
molecules dissociate, then it flattens out and becomes linear with a smaller 
slope as only slow molecules are still dissociating. So (in the absence of a 
curve-fitting program) you calculate the rate constant for the slow molecules 
from the linear region at long time, and extrapolate the line back to zero time 
to get the initial percent slow. Then you can calculate the amount of slow at 
any time point and subtract that from the total to get the amount of fast, and 
re-plot that on semilog to get the fast rate constant.

By analyzing our Wilson plots at 3A (or more generally at the highest 
resolution available) we are getting the B-factor for the slowest-decaying 
(lowest B-factor) atoms, getting B=10 not 13 in this case.





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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Increase in R-factor following REFMAC

[Schreuder, Herman /DE]

I guess the molecular replacement model has never been refined against this 
data set, so there is no reason why the Rfree should be better or worse than 
the Rfactor. The difference is larger than I would expect, but it may just be a 
statistical fluke. That the Rfree goes up significantly during refinement is 
what I would expect and suggests that the model needs significant rebuilding 
and refinement. Similar, the Rfactor going slightly up may indicate that the 
model is moving out of a false local minimum.

I would just continue rebuilding and refinement and see what happens.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Eleanor Dodson
Gesendet: Mittwoch, 8. April 2020 15:36
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Increase in R-factor following REFMAC


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Well - there is something funny about your first Rfree - it shouldnt be 
significantly lower than R?
I suspect there is some muddle over the assignment of Rfree - one used for 
Phaser and a different value for REFMAC?

And of course at low resolution especiall Rfactors are very sensitive to 
scaling procedures..
Eleanor

On Wed, 8 Apr 2020 at 14:08, Kyle Gregory 
<3632e92fcc15-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Hello,

I haven't seen this before but doing a round of refinment with REFMAC, after 
molecular replacement with phaser, my R factor and R free have increased?

Also is it weird that my Rfree is smaller than my R factor?

Result:
Initial
Final
R factor
0.3621
0.3761
R free
0.3108
0.4733
Rms BondLength
0.0076
0.0057
Rms BondAngle
1.6504
1.5193
Rms ChirVolume
0.0600
0.0526
My map was ok(ish) but there was indication of anistropy so I attempted to 
improve it by using STARANISO and re-ran phaser with the output, and this is 
the results I see following REFMAC.

Thanks,

Kyle



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] modelling MET (methionine) and SME (met-sulfoxide)

[Schreuder, Herman /DE]

Dear Abhishek,

I did not follow the links given by Paul. However the way I proceeded in these 
cases was to first generate an alternative conformation for the problem 
residues, save the file and then do the mutation and save the mutated file. 
Then, using an editor, I would cut the alternative conformation from the 
original residue and paste in the alternate conformation (e.g. conformation B) 
from mutated residue.

It is not very elegant, but it works (if coot and refmac cooperate).

Best, Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board  Im Auftrag von Abhishek Anan
Gesendet: Sonntag, 26. April 2020 21:42
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] modelling MET (methionine) and SME 
(met-sulfoxide)

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Dear all,

Thanks for the suggestions. It is synthetic peptide so the residue identity is 
unambiguous.

I am not clear on how to model both MET and SME in coot, do a real space 
refinement and then save the file for refinement in phenix. I tried alternate 
conformation and then mutating one of them but that didn't work as both 
conformations were mutated. What I also just noticed is that the refinement of 
structure with just SME results in positive densities around all side chain 
carbons even with the SME.cif loaded into phenix. What could be wrong here?

best wishes,
Abhishek





On 4/26/20, Paul Emsley  wrote:
>
> On 26/04/2020 16:21, Abhishek Anan wrote:
>> Dear all,
>>
>> I have a peptide crystal structure at 0.97 Å that contains two 
>> surface exposed Methionine. The CE atoms of both MET have a 
>> suspiciously high b-factor >40 and a positive density. In addition, 
>> the sulfur atom SD has a large negative density (b-factor ~23).
>>
>> I initially suspected that the MET may have oxidized to MET-sulfoxide 
>> and tried to model only the MET-sulfoxide. This again resulted in 
>> negative density.
>>
>> I think that the peptides might be partly oxidized which brings me to 
>> my question. Is there a way to model both MET and MET-sulfoxide into 
>> the density much like alternate conformation with options to refine 
>> their respective occupancies.
>
>
> Yes. This is called micro-heterogeneity
>
> And is documented here:
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.wwpdb.org_doc
> umentation_procedure&d=DwIFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5
> qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=CYQUM_mrfiCDADJ1
> onMNLQYYLwXD23pcMOTm7KnoNkM&s=A_ke05wSRD1nm9vQxFwLPCzpmUAWRTVlaVkVSTRw
> z8M&e=
>
> That should "just work" if you then give the model to refmac.
>
> FWIW, Coot is, AFAIR, not 100% happy with such models.
>
> Paul.
>
>
>



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[ccp4bb] AW: [ccp4bb] MR solution not working

[Schreuder, Herman /DE]

Hi Shubhashish,
How many molecules do you assume are in the asymmetric unit? You may have a 
very high solvent content and by trying to find multiple molecules, you spoil 
your solution. Also, did you do a thermal shift assay to make sure your protein 
is properly folded?

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Shubhashish 
Chakraborty
Gesendet: Donnerstag, 3. März 2022 05:44
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] MR solution not working

Hello,
I am trying to solve a dataset using molecular replacement. However, neither 
Phaser MR nor Molrep can give any solution.
In Phaser, I have received an advisory that Top FTF has not packed.
I have tried molecular replacement using the wild-type protein at different 
resolutions (I am working on a mutant).
Also, I have truncated the loops from the input structure. However, none have 
worked.
So, what can be the possible way to solve this data set?

Thank you

Shubhashish Chakraborty
PhD JRF 2018
Structural and Molecular Biology Lab (Varma Lab)
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Khargar, Navi Mumbai
E-mail: schakrabo...@actrec.gov.in



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[ccp4bb] AW: [ccp4bb] Ligand occupancy refinement

[Schreuder, Herman /DE]

Dear Ankanksha,

The ligand is either present, or it is not present. It cannot be that some 
atoms are present and others not. For ligands, I always use a single group 
occupancy using the program Buster from global phasing. In my hands, this 
always works. There is a correlation between occupancy and B-factors, but if 
you use a single occupancy for a large number of atoms, this correlation is not 
longer a problem. Also, modern maximum likelihood refinement programs do a good 
job in separating the contributions of occupancy and B-factors.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Jon Cooper
Gesendet: Donnerstag, 3. März 2022 16:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Ligand occupancy refinement

Hello, the ligand needs to be treated as one occupancy group since refining 
individual occupancies would be a case of refining too many parameters, unless 
it was a very fragmentary compound!! It is one keyword in refmac, but I can't 
remember for phenix, sorry! Ta jc


Sent from ProtonMail mobile



 Original Message 
On 3 Mar 2022, 14:15, Akanksha Tomar < 
akankshat...@gmail.com> wrote:

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing the protein 
model and water I fitted the ligand into it. Currently, I am using Phenix 
Refine with occupancy refinement for individual atoms switched on. After the 
refinement, the overall occupancy of the ligand is 0.7 and the RSCC value is 
0.86. The resolution of the structure is 2.1 Å.

Now the problem is that the program has assigned different occupancies to 
different atoms of the ligand. For some cases, it has assigned 0 occupancies to 
atoms for which there is a clear positive peak.

Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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[ccp4bb] AW: [ccp4bb] Ligand occupancy refinement

[Schreuder, Herman /DE]

PS: this does not work for very small atoms, e.g. waters. Here I let the 
temperature factor take care of the occupancy as well.

Von: Schreuder, Herman /DE
Gesendet: Donnerstag, 3. März 2022 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: AW: [ccp4bb] Ligand occupancy refinement

Dear Ankanksha,

The ligand is either present, or it is not present. It cannot be that some 
atoms are present and others not. For ligands, I always use a single group 
occupancy using the program Buster from global phasing. In my hands, this 
always works. There is a correlation between occupancy and B-factors, but if 
you use a single occupancy for a large number of atoms, this correlation is not 
longer a problem. Also, modern maximum likelihood refinement programs do a good 
job in separating the contributions of occupancy and B-factors.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Jon Cooper
Gesendet: Donnerstag, 3. März 2022 16:09
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] Ligand occupancy refinement

Hello, the ligand needs to be treated as one occupancy group since refining 
individual occupancies would be a case of refining too many parameters, unless 
it was a very fragmentary compound!! It is one keyword in refmac, but I can't 
remember for phenix, sorry! Ta jc


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 Original Message 
On 3 Mar 2022, 14:15, Akanksha Tomar < 
akankshat...@gmail.com<mailto:akankshat...@gmail.com>> wrote:

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing the protein 
model and water I fitted the ligand into it. Currently, I am using Phenix 
Refine with occupancy refinement for individual atoms switched on. After the 
refinement, the overall occupancy of the ligand is 0.7 and the RSCC value is 
0.86. The resolution of the structure is 2.1 Å.

Now the problem is that the program has assigned different occupancies to 
different atoms of the ligand. For some cases, it has assigned 0 occupancies to 
atoms for which there is a clear positive peak.

Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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[ccp4bb] AW: Determining space group

[Schreuder, Herman /DE]

Hi Monika,

I would process the data using the point group information ignoring any 
possible screw axes, e.g. in space group P222, where the true space group might 
be P212121.

The next step depends on how you plan to solve the structure. You mention that 
there is no cryo structure, do you mean cryoEM structure, or that you cannot 
freeze the crystals? If there is any search model available (crystal, cryoEM, 
alphaFold), I would run molecular replacement, using all possible space groups 
for your point group. This should resolve the correct space group.

Good luck!
Herman

Von: CCP4 bulletin board  Im Auftrag von Monika Bjelcic
Gesendet: Freitag, 22. Juli 2022 11:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Determining space group

Hi,

I’m hoping someone can help me how to determine a space group from my 
collection.
I did serial crystallography on a crystal that doesn’t have a cryo structure. I 
was able to determine the Point group but for the next step I’m stuck.

Kind regards,
Monika Bjelcic
PhD student at BioMAX
[cid:image001.jpg@01D89DC5.033B5D90]
MAX IV Laboratory
Lund University
Postal address: P.O. Box 118, SE-221 00 Lund, Sweden
Visiting address: Fotongatan 2, 224 84 Lund, Sweden
Mobile:  +46-761357994




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[ccp4bb] AW: [ccp4bb] Regarding the correct space group identification

[Schreuder, Herman /DE]

Dear Sayan,

If a subunit is correctly oriented, but the translation is incorrect, density 
for a ligand may still show up in the binding site of the protein. It might be 
that one of the 2-fold axes, you think is crystallographic, is in fact non 
crystallographic and a few Angstroms away from the crystallographic position.

What I would do:

  1.  Check the images: are there ice-rings or other artifacts that could cause 
scaling problems that would lead to high Rw/Rf values? In that case, there is 
not much you can do.
  2.  Compare the C2 and P22121 solutions: do they have the same overall 
crystal packing (CS+NCS), or are they different? Do they have the same Rw/Rf 
values? Can we learn anything from the differences in overall crystal packing?
  3.  Process, run MR and refine in P1. Do you get lower R-factors? If so, then 
run Zanuda to find out the real space group.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Sayan Saha
Gesendet: Donnerstag, 28. Juli 2022 08:15
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Regarding the correct space group identification

Dear All,

We have collected home-source X-ray intensity data for a protein at 2.6 
Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and 
beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained in 
both the space groups. However, the solution can be refined with an Rw/Rf of 
29/32% only. The protein is bound to a ligand (co-crystallization) for which a 
clear density can be observed.

Any help and suggestion in this regard would be very helpful.

With best regards,
Sayan Saha.




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[ccp4bb] AW: [ccp4bb] Regarding the correct space group identification

[Schreuder, Herman /DE]

Dear Sayan,

Thank you for this information. Why are your spots overlapping? The axes of 
your crystal are not particularly long. Did you put the detector very close to 
the crystal, or are there multiple diffraction patterns?

Did you run Zanuda on your P1 structure? What Rfactors do you get when you 
complete the refinement in P1?

Best regards,
Herman

Von: Sayan Saha 
Gesendet: Donnerstag, 28. Juli 2022 11:43
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Regarding the correct space group identification

Dear Sir,

1. There are no ice-rings. However, diffraction spots seem to be overlapping. 
This can be seen during the data processing, as the space group (C2 or P222) 
varies even in the consecutive frames.

2. Crystal packing of C2 and P22121 seem to be similar (please see the attached 
images).

3. Forgot to mention in my previous email that we have already processed the 
data in P1 and MR solution could be found only in P1 (Phaser was used with an 
option in all possible space groups of that point group).

Please let me know if any other information is required.

With best regards,
Sayan Saha.


On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Sayan,

If a subunit is correctly oriented, but the translation is incorrect, density 
for a ligand may still show up in the binding site of the protein. It might be 
that one of the 2-fold axes, you think is crystallographic, is in fact non 
crystallographic and a few Angstroms away from the crystallographic position.

What I would do:

  1.  Check the images: are there ice-rings or other artifacts that could cause 
scaling problems that would lead to high Rw/Rf values? In that case, there is 
not much you can do.
  2.  Compare the C2 and P22121 solutions: do they have the same overall 
crystal packing (CS+NCS), or are they different? Do they have the same Rw/Rf 
values? Can we learn anything from the differences in overall crystal packing?
  3.  Process, run MR and refine in P1. Do you get lower R-factors? If so, then 
run Zanuda to find out the real space group.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Sayan Saha
Gesendet: Donnerstag, 28. Juli 2022 08:15
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] Regarding the correct space group identification

Dear All,

We have collected home-source X-ray intensity data for a protein at 2.6 
Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and 
beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained in 
both the space groups. However, the solution can be refined with an Rw/Rf of 
29/32% only. The protein is bound to a ligand (co-crystallization) for which a 
clear density can be observed.

Any help and suggestion in this regard would be very helpful.

With best regards,
Sayan Saha.




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[ccp4bb] AW: [ccp4bb] Regarding the correct space group identification

[Schreuder, Herman /DE]

Dear Sayan,

your options left, as far as I can see, are:

- check if your structure is not twinned, or if not, correct for twinning
- refine using the Zanuda space group.
- try to find a better crystal that does not produce multiple diffraction 
images.

Best,
Herman

Von: Sayan Saha 
Gesendet: Donnerstag, 28. Juli 2022 15:17
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Regarding the correct space group identification

Dear Sir,
 The crystal-to-detector distance was set to 190 mm. Yes, multiple diffractions 
seem to be present. We have not yet tried Zanuda on the P1 structure. However, 
the Rw/Rf of P1 structures are little higher (31/34%).

With best regards,
Sayan Saha.

On Thu, Jul 28, 2022 at 5:22 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Sayan,

Thank you for this information. Why are your spots overlapping? The axes of 
your crystal are not particularly long. Did you put the detector very close to 
the crystal, or are there multiple diffraction patterns?

Did you run Zanuda on your P1 structure? What Rfactors do you get when you 
complete the refinement in P1?

Best regards,
Herman

Von: Sayan Saha mailto:ssaha43...@gmail.com>>
Gesendet: Donnerstag, 28. Juli 2022 11:43
An: Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: Re: [ccp4bb] Regarding the correct space group identification

Dear Sir,

1. There are no ice-rings. However, diffraction spots seem to be overlapping. 
This can be seen during the data processing, as the space group (C2 or P222) 
varies even in the consecutive frames.

2. Crystal packing of C2 and P22121 seem to be similar (please see the attached 
images).

3. Forgot to mention in my previous email that we have already processed the 
data in P1 and MR solution could be found only in P1 (Phaser was used with an 
option in all possible space groups of that point group).

Please let me know if any other information is required.

With best regards,
Sayan Saha.


On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Sayan,

If a subunit is correctly oriented, but the translation is incorrect, density 
for a ligand may still show up in the binding site of the protein. It might be 
that one of the 2-fold axes, you think is crystallographic, is in fact non 
crystallographic and a few Angstroms away from the crystallographic position.

What I would do:

  1.  Check the images: are there ice-rings or other artifacts that could cause 
scaling problems that would lead to high Rw/Rf values? In that case, there is 
not much you can do.
  2.  Compare the C2 and P22121 solutions: do they have the same overall 
crystal packing (CS+NCS), or are they different? Do they have the same Rw/Rf 
values? Can we learn anything from the differences in overall crystal packing?
  3.  Process, run MR and refine in P1. Do you get lower R-factors? If so, then 
run Zanuda to find out the real space group.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Sayan Saha
Gesendet: Donnerstag, 28. Juli 2022 08:15
An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Betreff: [ccp4bb] Regarding the correct space group identification

Dear All,

We have collected home-source X-ray intensity data for a protein at 2.6 
Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and 
beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained in 
both the space groups. However, the solution can be refined with an Rw/Rf of 
29/32% only. The protein is bound to a ligand (co-crystallization) for which a 
clear density can be observed.

Any help and suggestion in this regard would be very helpful.

With best regards,
Sayan Saha.




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[ccp4bb] AW: [ccp4bb] Regarding the correct space group identification

[Schreuder, Herman /DE]

Dear Sayan,

There are no multiple diffraction patterns in the images you sent us. However, 
the mosaicity appears to be quite high and in this case I would really 
recommend to process the data with XDS (if you have not already done that).

In the left side of image 2, there appear to be alternating loons with strong 
single spots, and loons with rows of streaky spots which are very close 
together, which reminds me of a crystal with lattice translocation disorder I 
once struggled with. However, it might also be bona fide diffraction, with the 
streaky spots caused by a combination of your long cell axis with high 
mosaicity.

You can check this by overlaying the predicted spots on your diffraction 
pattern. All data processing programs have an option to do this.

Finally, I would superimpose your MR solutions of C2 and P222 and look if they 
are exactly the same. If they are different, e.g. if one of the subunits in one 
of the space groups is translated with respect to the other, your crystal may 
contain a mixture both, causing to problems you are facing.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Sayan Saha
Gesendet: Donnerstag, 28. Juli 2022 18:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Regarding the correct space group identification

Dear Sir,

The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
degree. Please find attached two diffraction images.
With best regards,
Sayan Saha.


On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs 
mailto:kay.diederi...@uni-konstanz.de>> wrote:
Dear Sayan,

On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha 
mailto:ssaha43...@gmail.com>> wrote:

>Dear Sir,
>
>1. There are no ice-rings. However, diffraction spots seem to be
>overlapping. This can be seen during the data processing, as the space
>group (C2 or P222) varies even in the consecutive frames.

spot overlap results in inaccurate intensity values. Inaccurate intensities 
result in high Rwork/Rfree.

Why do the spots overlap? High mosaicity? Detector distance too small? 
Oscillation range too high (0.1° is typically adequate)?

It would be good to see the data, otherwise we can only speculate.

Space group does not change from one frame to the next. If you use XDS, a good 
guide to decide between higher and lower-symmetry space groups is to compare 
their ISa values.

best,
Kay

>
>2. Crystal packing of C2 and P22121 seem to be similar (please see the
>attached images).
>
>3. Forgot to mention in my previous email that we have already processed
>the data in P1 and MR solution could be found only in P1 (Phaser was used
>with an option in all possible space groups of that point group).
>
>Please let me know if any other information is required.
>
>With best regards,
>Sayan Saha.
>
>
>On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE <
>herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> wrote:
>
>> Dear Sayan,
>>
>>
>>
>> If a subunit is correctly oriented, but the translation is incorrect,
>> density for a ligand may still show up in the binding site of the protein.
>> It might be that one of the 2-fold axes, you think is crystallographic, is
>> in fact non crystallographic and a few Angstroms away from the
>> crystallographic position.
>>
>>
>>
>> What I would do:
>>
>>1. Check the images: are there ice-rings or other artifacts that could
>>cause scaling problems that would lead to high Rw/Rf values? In that case,
>>there is not much you can do.
>>2. Compare the C2 and P22121 solutions: do they have the same overall
>>crystal packing (CS+NCS), or are they different? Do they have the same
>>Rw/Rf values? Can we learn anything from the differences in overall 
>> crystal
>>packing?
>>3. Process, run MR and refine in P1. Do you get lower R-factors? If
>>so, then run Zanuda to find out the real space group.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> *Von:* CCP4 bulletin board 
>> mailto:CCP4BB@JISCMAIL.AC.UK>> *Im Auftrag von *Sayan
>> Saha
>> *Gesendet:* Donnerstag, 28. Juli 2022 08:15
>> *An:* CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
>> *Betreff:* [ccp4bb] Regarding the correct space group identification
>>
>>
>>
>> Dear All,
>>
>>
>>
>> We have collected home-source X-ray intensity data for a protein at 2.6
>> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and
>> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained
>> in both the space groups. However, the solution can be refined with an
>> Rw/Rf of 29/32% only. The protein is bound to a ligand (co-crystallizatio

[ccp4bb] AW: [ccp4bb] Making bonds

[Schreuder, Herman /DE]

Dear Cryo EM,

In general, if you bring the two atoms at approximatively the correct distance 
and do a real space refinement or regularization, a bond should be generated.
However, there are caveats:

  1.  Do the nucleotides belong to the same chain? If they belong to different 
chains, they will not get bonded.
  2.  Do the atoms have the correct names for standard nucleotides?
  3.  An erroneous TER card between the nucleotides may also behind your 
problem.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Cryo EM
Gesendet: Dienstag, 9. August 2022 03:41
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Making bonds

Hi everyone,

In one my PDBs, I see that there is a linkage break in ribonucleotides. 
Specifically one of the phospodiester bond seems to be broken and thus O3' 
needs to be bonded to P atom of next nucleotide. What is the easiest way to do 
this in coot ? Or any other software?
I tried "make link" option in coot but it seems it just created link in PDB and 
not bond?


Regards




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[ccp4bb] AW: Help with isomer ligand from PDB database

[Schreuder, Herman /DE]

Dear Marion,

If you read in your corrected .cif file in Coot or your refinement program, it 
will override the (default) CCP4 .cif file. You can also correct the installed 
CCP4 .cif file. I once did this. However, these changes are lost when you 
update the CCP4 installation, so best is to ask the CCP4 developers do correct 
their .cif file.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Marion Schuller
Gesendet: Samstag, 17. September 2022 16:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Help with isomer ligand from PDB database


Dear CCP4bb community,

I am writing regarding a problem with the refinement of an isomer ligand. I try 
to refine a structure which has the ligand "Carba-NAD" (as beta isomer) bound. 
This ligand is already in the pdb database as "CNA". Although the ligand is in 
its beta-Carba-NAD form in the database, it is loaded into WinCoot as alpha 
isomer. When generating the restraints of Carba-NAD (via the grade server, 
calling it also "CNA"), I can load and refine the correct beta isomer ligand as 
long as I provide the grade-generated .cif file otherwise it would refine it 
back to the alpha isomer. Yet, in the preliminary preparation of the wwPDB 
X-ray validation report for structure deposition, the message flags up that the 
ligand "could not be matched to an existing wwPDB Chemical Component Dictionary 
definition". With an older ccp4 version in the background, I noticed that 
WinCoot would however load the beta isomer and I am now wondering if this 
problem lies in the .cif file of the new ccp4 library. Is there a possibility 
to check/correct the CNA file in the current ccp4 library or would there be a 
different way for solving this problem? I would be very grateful for support 
and help!

With kind regards and many thanks in advance,
Marion




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[ccp4bb] AW: Multiplicity is more than 20

[Schreuder, Herman /DE]

Hi Prasun,

As others have pointed out, the higher the multiplicity, the better, so I would 
be happy about it and not worry.
Concerning the higher Rwork in the inner shell, what are the resolution limits 
of this shell? Are there any ice-rings within these resolution limits? 
Otherwise I would check if there are no overloaded reflections in your data 
set, since a few rogue strong reflections may wreak havoc on your Rfactors.

Best, Herman

Von: CCP4 bulletin board  Im Auftrag von Prasun Kumar
Gesendet: Montag, 19. September 2022 20:30
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Multiplicity is more than 20

Hi All:

I have collected a dataset for a crystal of a 30 residues long helical peptide 
that makes a trimer in the solution. I also solved the structure to get a 
trimer. My issues start when I start preparing for a deposition.

Details about the data:

space group: I 21 3
Resolution: 1.6
Current Rfree/ Rwork: 0.21/0.19

Problems:
According to Aimless, Multiplicity: 33.9, and I understand that the value 
should be less than or equal to 20. Does it mean that I have a lot of random 
noise or ice rings or something similar?
For the inner shell, R work is also higher than R free.

Please guide me in solving the above issue.

Thank You
Prasun




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[ccp4bb] AW: [ccp4bb] PAIREF, Anisotropy and STARANISO

[Schreuder, Herman /DE]

Dear Martin,

The concept of systematic absences is usually used for absences caused by the 
crystal packing, e.g. glide planes etc. I guess that what you meant are 
absences due to anisotropic cutoff. In the past, to get an Rmerge is low as 
possible, people would cut there data at 2sigma, or even at 3 sigma, resulting 
in data sets with significant high-resolution data missing. The same happened 
if the detector was placed to far away from the crystal.

If the anisotropic cutoff is done correctly, e.g. at 1sigma or lower, the 
measured data set is complete. The fourier terms that are missing do not have 
any significant amplitude and are basically noise. So if all refinement and 
other software would be perfect, there would be no artefacts due to missing 
data, since there is no missing data, only missing noise. In your words, the 
missing data is extremely weak, not strong.

However, and I guess this is what Frank was hinting at, this anisotropic 
diffraction should be correctly handled by the refinement programs and should 
be modeled correctly in the resulting models. A first step would be matching 
overall anisotropic B-factors but I am sure there are more sophisticated 
solutions.

My 2 cents,
Herman

Von: CCP4 bulletin board  Im Auftrag von Martin Malý
Gesendet: Mittwoch, 5. Oktober 2022 14:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] PAIREF, Anisotropy and STARANISO

Dear colleagues,

Firstly, I would like to thank Clemens, Claus, Ian and Gerard for the very 
interesting analysis and remarks!

Similarly to Frank, I am wondering "whether any refinement program properly 
extracts the high-resolution signal when there's anisotropy". Poor completeness 
in high resolution can play a role. Somewhere in the "gear wheels" of 
refinement programs (refmac5, phenix.refine, buster, ...), Fourier transform 
has to take place. It is a sum through hkl indices which somehow assumes 
isotropic data to "work reasonably", I think. However, anisotropic cutoffs 
produce strong systematic absences in data. The refinement programs must fit 
the structure model on significantly incomplete data, which is quite 
questionable. On the other hand, structure refinement is usually stable and 
converges also when using anisotropically-cut data so that is a good sign.

To conclude, I am still wondering if there should or should not be a criterion 
for the minimal spherical completeness in high resolution. This would probably 
vary for particular refinement programs/strategies.

Best regards,
Martin


On 05. 10. 22 11:36, Frank von Delft wrote:
Gerard, that's fascinating, thanks for the explanation.

I conclude in summary:  nobody (including you) yet knows whether any refinement 
program properly extracts the high-resolution signal when there's anisotropy.

My (similar) question a few days ago was: "Is that because of the 
practicalities of implementing the numerical methods, or because of something 
fundamental about the refinement formalism?"

I think you say below that the problem is (might be?) that the error models and 
corrections assume isotropic behaviour.

So if that doesn't work, shouldn't the error models instead be derived from the 
statistics of local reciprocal space?  As you do in STARANISO (from memory).

Frank






On 04/10/2022 17:01, Gerard Bricogne wrote:

Dear all,



 First of all, apologies for breaking the threads entitled "PAIREF -

Warning - not enough free reflections in resolution bin" and "Anisotropy" by

merging them into a new one, but it somehow felt rather against nature to

keep them separate.



 Since the early days of the availability of STARANISO [1] (the actual

starting year for the Web server [2] was 2016), we had a hunch that much of

what was happening in the PAIREF procedure might simply be the detection of

the existence of significant data beyond an initially chosen resolution

cut-off not only as a result of an excessively conservative criterion having

been applied in that initial choice, but as a consequence of anisotropy in

the data. The latter would give rise to different diffraction limits in

different directions, and the choice of a single value for "the resolution"

at which the data were cut off would necessarily yield a compromise value

between the best and the worse diffraction limits. This would imply that

significant data would be excluded in the best diffracting directions, that

would subsequently drive PAIREF towards increasing the estimated resolution

compared to its compromise value.



 This "hunch" was validated by a detailed comparison carried out on the

exact same examples that are considered in the 2020 paper by Maly et al.,

that is summarised in the attached PDF. In other words, whenever anisotropy

is present in the data, PAIREF will tend to indicate a higher value for an

isotropic cut-off than would have been estimated for the initial dataset.

The problem with taking the PAIREF result as the final answer is that the

higher cut-off 

[ccp4bb] AW: [ccp4bb] Fragmented Chains after Automated Rebuilding

[Schreuder, Herman /DE]

Dear Shawn,

I am not aware of an automated way to merge all fragments into a single 
consistent peptide chain. What I would do is to look at the map and the built 
fragments in coot and switch the symmetry on with a large radius, e.g. 20-30A 
and see if you can find a set of fragments (including symmetry mates) that 
could form a complete and consistent protein molecule.

If there is a suitable MR model available, I would try to match the fragments 
to the model (find equivalent amino acids). You could use the matching residues 
to superimpose the MR model e.g. onto the largest fragment available. The next 
step would be to rebuild the MR model using the electron density map and the 
built fragments as a guide.

If there is no suitable MR model, I would probably built the whole protein, 
starting from the largest fragment, using the built residues as a guide. For 
this you can use the “add residue” option and put the residue created at the 
position of the equivalent residue of a fragment. Every 3-5 residues, you 
should do a real space refinement. The pdb standard accepts negative residue 
numbers, so you should be able to build in N-terminal direction as well.  
Afterwards you will have to renumber the protein to get the correct numbering.

The alternative would be to select the correct symmetry mate for each fragment, 
write them out, renumber them and merge them into a single pdb file. However, 
in my experience, this is much more hassle than the build them “from scratch” 
as I mentioned.

Good luck,
Herman



Von: CCP4 bulletin board  Im Auftrag von Shawn Rumrill
Gesendet: Mittwoch, 5. Oktober 2022 22:42
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Fragmented Chains after Automated Rebuilding

Hello All,

I am trying to solve a protein/nucleic acid (na) structure. The density for na 
is poor and MR is less straightforward, so I used ModelCraft for automated 
phasing, rebuilding and improving the electron density. It has built a partial 
model for the complex, which looks somewhat reasonable, however, the protein 
and na chains are fragmented, which throws off numbering, etc. I'm sure this is 
expected, but I typically don't do automated rebuilding (necessary in this 
case).

Is there any way to easily connect and renumber the chains and residues 
(perhaps using a reference PDB structure) to try and build something more 
complete (in terms of chains and residues)?

I have not worked with a protein/na complex so certainly na modeling is new to 
me. Any input is greatly appreciated!

Thanks for your expertise,

Shawn



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[ccp4bb] AW: [ccp4bb] mutate Methionine to Norleucine in Coot.

[Schreuder, Herman /DE]

Hi Jiang,
It could have a lot of causes. What I would first check is that the chainID of 
the NLE is the same as from the surrounding protein, that the new NLE has the 
same residue number as the deleted MET. I would also check that the position 
within the coordinate file is the same as the previous MET.

Good luck!
Herman

Von: CCP4 bulletin board  Im Auftrag von Jiang Xu
Gesendet: Donnerstag, 20. Oktober 2022 03:07
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] mutate Methionine to Norleucine in Coot.

Hi Garib,
   I deleted the Met residue and imported the nle.cif file into the dictionary 
and checked the "create a molecule" box.I then used "real space refinement" to 
align it correctly with the electron density. I then save the merged molecule 
as a new pdb file but when I reopened the new file, I found the NLE molecule's 
position was not aligned with the electron density and couldn't be corrected 
with "real space refinement" in Coot. So any suggestions?
Thank you,
Best,
Jiang

On Wed, Oct 19, 2022 at 12:44 PM Garib Murshadov 
mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:
NLE is in the monomer library. However, it is marked as non-polymer. The reason 
why it is non-polymer is that one of the hydrogen atoms on N is called HN2 (in 
peptides they are H, H2 and H3).
One easy way would be replace HN2 with H and save in a cif file. Then read it 
in coot using “import cif dictionary" and hope that coot will recognise it as 
peptide. I attach nle.cif just in case

Regards
Garib



On 19 Oct 2022, at 20:34, Jiang Xu 
mailto:foxj...@gmail.com>> wrote:

Hello Guys,
   I have a question regarding how to change the standard amino acid in my 
structure to Norleucine. It turned out that the one Methionine should be 
Norleucine. I tried to use the coot's mutation method but didn't find 
NLE(Norleucine) there. Any suggestions?
Thank you,
Best,
Jiang
Lin Chen Lab
University of Southern California




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[ccp4bb] AW: [ccp4bb] Low resolution and high anisotropy

[Schreuder, Herman /DE]

Dear Shenyuan,

I have two suggestions:

  1.  Is your protein a single or a multi-domain protein? If it is a 
multi-domain protein, you should search with the individual domains.
  2.  Given the low resolution, high anisotropy and expected large number of 
molecules in the asymmetric unit, you may have an exceptionally high solvent 
content, maybe >75%. Is the electron density of all molecules bad, or have a 
few molecules better electron density? If a few molecules have reasonable 
density, I would keep those and delete the molecules with bad density. 
Alternatively, I would start a MR search for only 3 or 4 molecules.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Xu, Shenyuan
Gesendet: Montag, 24. Oktober 2022 03:43
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Low resolution and high anisotropy

Dear CCP4 community,

I have encountered a dataset, which I thought should be easy to solve. The 
volume of the cell unit seems to be expanded after image 271, which I think is 
caused by radiation damage. After removing the last few images, the scaled 
statistics seem good with the resolution set at 3:07 A:

d_max  d_min   #obs  #uniq   mult.  %comp r_mrg   r_meas   
 r_pim   r_anom   cc1/2   cc_ano
 91.54   8.33   3086   11972.58  98.44 345.251.30.1010.133  
  0.0860.145   0.973*  -0.148
  8.33   6.61   3005   12252.45  99.51 256.527.30.1600.212  
  0.1380.260   0.923*  -0.140
  6.61   5.78   2883   11902.42  98.43  92.9 9.40.2780.368  
  0.2370.461   0.541*  -0.165
  5.78   5.25   3195   12172.63  99.10  75.6 8.10.2830.369  
  0.2330.436   0.618*  -0.107
  5.25   4.87   3192   12062.65  99.42  92.5 8.30.2810.367  
  0.2320.427   0.856*  -0.187
  4.87   4.59   3230   12132.66  99.02 118.410.30.2880.378  
  0.2400.456   0.870*  -0.053
  4.59   4.36   2797   11562.42  92.93 137.012.20.3090.410  
  0.2660.496   0.843*  -0.239
  4.36   4.17   2694   11182.41  93.17 280.216.20.4190.575  
  0.3920.847   0.491*  -0.142
  4.17   4.01   3102   11882.61  95.50 133.6 9.90.3920.516  
  0.3300.622   0.663*  -0.268
  4.01   3.87   3224   12052.68  98.69 120.4 7.30.4060.539  
  0.3490.687   0.791*  -0.126
  3.87   3.75   2939   11812.49  98.91  90.7 6.80.4910.652  
  0.4250.810   0.691*  -0.042
  3.75   3.64   1981   10211.94  82.34  87.3 5.00.5570.756  
  0.5060.885   0.540*  -0.018
  3.64   3.54   2374   10822.19  89.13 194.611.20.5020.675  
  0.4470.973   0.625*  -0.237
  3.54   3.46   2622   11222.34  92.35 310.510.10.4320.585  
  0.3920.851   0.603*  -0.154
  3.46   3.38   17399451.84  76.58  48.2 3.60.9301.247  
  0.8221.542   0.444*  -0.046
  3.38   3.31   2847   12402.30  98.57  85.4 4.20.6910.930  
  0.6161.242   0.523*  -0.060
  3.31   3.24   2838   11532.46  97.88  70.8 3.30.6930.934  
  0.6201.341   0.362*   0.006
  3.24   3.18   3097   12122.56  97.66  71.4 2.50.6920.924  
  0.6051.173   0.526*   0.044
  3.18   3.12   3204   12162.63  99.10  79.6 3.70.6680.886  
  0.5761.338   0.440*  -0.081
  3.12   3.07   3059   11722.61  98.16  73.5 2.40.7140.945  
  0.6121.633   0.362*  -0.177
 91.50   3.07  57108  232592.46  95.22 138.410.80.3830.513  
  0.3360.671   0.656*  -0.149

I used Mrbump to do the MR, most sequence identities of the starting templates 
are more than 0.85, and some of them are structures predicted from alpha fold 
2. But after refinement (including jelly-body, proSmart, TLC), the best R/Free 
R stuck at around 0.42/0.45. Inspecting the electron density map shows that the 
model does not fit the electron density well. The space group is P1, Cell is 
58.49 63.97 91.60 91.71 91.86 99.47, should be 5 or 6 molecules in the 
asymmetric unit.

I checked the data quality, it said the data is highly anisotropy. Then
I searched the CCP4 forum and used the STARANISO Server and UCLA server, but 
still cannot improve the refinement. The data statistics after drawing 
ellipsoidal resolution limits is good:

 
 resolution   observed redundancycompletenesrmerge 
i/sigma
before/after  before/after   before/afterbefore/after   
before/after
7.90 4957  1185 3.5  3.397.3%   92.6%13.0%  12.7%11.9   
7.2
5.58 8023  2397 3.0  3.698.6%   98.9%30.3%  11.8% 5.5   
7.5
4.5611176  2624 3.3  3.199.1%   97.5%57.1%  18.2% 3.7   
6.3
3.9511694  3213 2.9  3.194.0%   99.0%53.1%  27.5% 4.2   
4.3
3.5312065  3524 2.6  3.092.8%   99.0%49.8%  45.9% 3.2   
2.8
 

[ccp4bb] AW: [ccp4bb] occupancy factors for alternate conformations and alternate ligands

[Schreuder, Herman /DE]

Hi Mike,
My gut feeling is that the accuracy will be around 10%. However it will depend 
very much how far the two conformation overlap, or not overlap and how good the 
data is. If there are large non-overlapping regions, accuracy may be better.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Harry Powell
Gesendet: Dienstag, 29. November 2022 13:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] occupancy factors for alternate conformations and 
alternate ligands

Hi MIke

Beyond the obvious that they should sum to no more than unity, if they are 
similar to or not hugely greater than other temperature factors in your 
structure, they are probably okay.

People often apply restraints or constraints to keep the temperature factors 
similar for equivalent atoms in different partially occupied conformations, but 
I’m not sure there is a real justification for this apart from making the 
solution refine more stably. No doubt someone here will correct me!

Harry

> On 28 Nov 2022, at 19:31, Michael Colaneri 
> mailto:colane...@oldwestbury.edu>> wrote:
>
> Dear colleagues,
> If I have alternate ligands overlapping in the same electron density or 
> alternate conformations of the protein chain overlapping in the same electron 
> density, how accurate are the occupancy factors? (Res 2A - temp factors are 
> fine)
> Thanks, Mike Colaneri
>
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