[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan complex

[Herman . Schreuder]

Hi Shen,

I agree with Eleanor that the split spots will cause worse statistics, but 
should not be a reason for molrep to fail.
What I would do:
Molecular replacement with a resolution cut of 3 or 3.5 Å.
Process and run molecular replacement in P1. You may have some tricky 
pseudo-symmetry. With 8 molecules, molrep may take somewhat longer, but I think 
it is worth a try.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Xu, Shenyuan
Gesendet: Montag, 11. November 2019 16:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan complex


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

Hello Eleanor,

I used the solved one as the input model. The space group of the solved 
crystals are: P1 21 1, and P 21.

I will turn the anomalous processing off.

Thanks,

Shen

On Mon, Nov 11, 2019 at 10:40 AM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Why dont you use one of your solved structures as a model?
What space group are the solved ones in?
The split spots are not good, but they are well separated and integration 
programs are not bad at such spacings? And your R merges etc look OK.
Just a Q - why turn off the anomalous processing. You dont need to use it, but 
at some point it may help you find Ss or Ca or whatever..
Eleanor


On Mon, 11 Nov 2019 at 15:24, Xu, Shenyuan 
mailto:x...@miamioh.edu>> wrote:
Dear All,

Thanks for all of your nice suggestions! (Sorry I did not respond to individual 
email to say Thanks.) I have used phaser to go through all possible space 
groups in the same point group(choose "all possible in same pointgroup" in the 
space group menu). I also try P43212 alone. The best result I get from phaser 
is (Top LLG: 26.490, Top TFZ: 6.7, Spacegroup: P41 2 2), and refinement ends in 
the R-work: ~0.54, R-free:~0.57.

I go back to see the diffraction data, and it seems that spots in the high 
resolution bin are severely split (pictures are attached). I feel like the fail 
of replacement is caused by this. Is this caused by twinning?

I am looking forward suggestions!

Thanks,

Shenyuan Xu

On Wed, Nov 6, 2019 at 2:58 PM Kay Diederichs 
mailto:kay.diederi...@uni-konstanz.de>> wrote:
Dear  Shenyuan,

it seems you assumed P41 21 2 is correct but it isn't - did you try P43 21 2 in 
molecular replacement?

The pointless log file mentions this alternative a few lines above the ones 
you've quoted.

If that does not work, try the other P4x 2y 2 space groups. All of this can be 
done in a single phaser run (choose "all possible in same pointgroup" in the 
space group menu).

Why did you cut the data at 2.29A? They are very strong in the high resolution 
shell!

HTH,
Kay

On Wed, 6 Nov 2019 14:29:13 -0500, Xu, Shenyuan 
mailto:x...@miamioh.edu>> wrote:

>Dear all,
>
>I am working on a protein-glycan complex trying to use molecular
>replacement.This protein is a VP8* domain from rotavirus, adopting a
>classical galectin-like fold. I already used MR to solve some other strains
>(apo-form and complex form). However, When I want to use the same model to
>solve this particular strain (sequence identity > 95%), MR seems to be
>failed with R=0.54, Free R = 0.57, and the model does not agree with the
>electron density map once viewed on coot. One difference of this
>protein-glycan complex between others is that the glycan is longer, which
>has six carbohydrate unit.
>
>I used imosflm trying to reindex and use pointless to check the space
>group, it usually ends up with the current choice:
>25_pointless.log
>
>WARNING! one or more zones have data systematically missing from the input
>file
>thus we cannot determine if reflections are truly systematically absent
>
>Best Solution: space group P 41 21 2
>Reindex operator: [h,k,l]
>Laue group probability: 1.000
>Systematic absence probability: 0.995
>Total probability: 0.995
>Space group confidence: 0.993
>Laue group confidence 1.000
>
>WARNING: You will have to resolve the enantiomorphic ambiguity later
>
>Unit cell: 106.78 106.78 138.59 90.00 90.00 90.00
>
>84.58 to 2.43 - Resolution range used for Laue group search
>
>84.58 to 2.29 - Resolution range in file, used for systematic absence check
>
>Number of batches in file: 1
>
>The data do not appear to be twinned, from the L-test
>I also use Zanuda to validate the space group, it ends with the same choice:
>
>Step 3.
>   Refinement of the best model.
>   Candidate symmetry elements are added one by one.
>
>   current time:Nov 06 16:17 GMT
>   expected end of job: Nov 06 17:06 GMT
>
>   ^
>   | >>   3   | C 1 2 1|  0.3736  |  0.5334  |  0.5044  |  0.5586  |
>   -
>   |  1   | P 1|  0.4058  |  0.5318  |  0.5020  |  0.5624  |
>   |  5   | P 1 21 1   |  0.4928  |--|  0.4973  |  

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Dear HK,

I agree with Artem, I would put the ketone in the green density below in your 
pictures and make a covalent link with the arginine. The single oxygen I would 
put in the density currently occupied by the ketone and add one water there as 
well. In your refined omit maps, the current ketone density is rather weak and 
weakly connected with the ligand. This is more reminiscent of a bound water 
than of a covalent part of the ligand.

Best,
Herman

-Ursprüngliche Nachricht-
Von: t...@em.uni-frankfurt.de  
Gesendet: Donnerstag, 7. November 2019 13:51
An: Dale Tronrud ; Seijo, Jose A. 
; Schreuder, Herman /DE 

Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de   



Dear Dale, Jose, Herman,

For Dale,
The contour level of residual positive density in the map is 0.28
e/A^3 and 3 sigma as shown in Coot, when it is refined in full occupancy. I 
tried to model the amino-sugar (flip the ligand) to the mysterious density but 
it has no space for it as it clashes with the surrounding amino acids.

For Herman,
I noticed that there are some impurities of the ligand from the literature and 
it also mentions that the ligand tends to be degraded due to pH and oxidation. 
It might be a degraded product.

For Jose,
I tried to refine it with one water molecule instead of two (Figure - 
fullocc1.png and fullocc2.png. They are the same but with different
view) and positive density seems to be reduced with full occupancy refinement 
of ligand and water. I also performed a refinement with occupancy refinement 
with ligand and water
(occupancy_ligand_water.png) but it seems to me that positive density appears 
again in comparison with full occupancy refinement of water.  
But the B-factor of water is too low if refine it with a occupancy refinement. 
If occupancy of water is set to 1 then the B-factor seems to be correlated well 
with the surroundings.

Thanks

Best
HK


Quoting Dale Tronrud :

> Hi,
>
>I'm curious.  What is the contour level of the residual positive 
> density in the maps with full occupancy ligand and Arg, and how does 
> that level compare to the level of the contour of your omit map?  
> e/A^3 would be the most useful.
>
>Without the contour levels it isn't possible to distinguish between 
> the absence of fully occupied atoms and something more minor.
>
>Could your ligand be spending a small part of its time flipped over 
> on the long axis of your fused ring system?  This would put the 
> sugar-like group over by your mystery density.
>
> Dale Tronrud
>
> On 11/6/2019 10:58 PM, Heng-Keat Tam wrote:
>> Hi Herman,
>>
>> Thanks for the suggestion. In principle, I have refined the structure 
>> without Arg and ligand, as well as the connected residues to the Arg.
>> However, I still see the connected density of Arg and the ligand 
>> (Figure
>> - unmodel1.png - only removal of Arg and ligand).
>>
>> I have tried to refine the degraded product but the positive density 
>> in between Arg and the ligand is still there, indicating something 
>> should be modeled there (Figure - degraded_product.png and 
>> degraded_product2.png - both figures are the same ligand but with 
>> different side view).
>>
>> I also tried to model the ligand in different conformation but still 
>> the same as shown in degraded product. The positive density is there 
>> between Arg and the ligand.
>>
>> For both cases, I refined Arg with full occupancy but ligand with 
>> occupancy refinement. Now, Arg did not flip away from the density.
>>
>> I might have a look at the mass-spec to check whether it is 
>> covalently linked although I am not aware of the reactivity of the 
>> moiety of the ligand and we never see this compound to covalent link 
>> with protein in our experience.
>>
>> Thanks for the advice and suggestion. It is very helpful.
>>
>> Thanks.
>>
>> Best
>> HK
>>
>>
>>
>> Quoting herman.schreu...@sanofi.com:
>>
>>> Hi Heng-Keat,
>>>
>>> I had again a look at your electron density pictures and these were 
>>> my
>>> impressions:
>>> 1) the Arginine as fitted looks fully occupied. There are no hints 
>>> for an alternative conformations.
>>> 2) The fit of the ligand, although reasonable, is not extremely good.
>>>
>>> It seems that the only reason to invoke partial occupancies for the 
>>> Arginine and ligand are problems to fit both the ligand and the 
>>> arginine in the available density. This means that for the 
>>> overlapping parts, to sum of the occupancies is maximal 1.0. 
>>> However, the ligand and the Arginine do not look half occupied.
>>>
>>> So I would look at other explanations:
>>> - would it be possible that the ligand reacts covalently with the 
>>> Arginine? The density looks pretty continuous to me.
>>> - could it be that instead of the intended ligand, some side- or 
>>> degradation product has bound?
>>>
>>> What I would also 

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Dear HK,

In your case mass spec would be extremely valuable. Not only could it prove or 
rule out a covalent link, it will also convince referees that the link is real.
There is clearly more density than the degraded product you showed. I would 
also ask an experienced chemist if a link between your ligand and the arginine 
would be possible. Finally, I would also do a quality check on the ligand you 
used. Might it be that some reactive intermediate from the synthesis still is 
present?

Best,
Herman

-Ursprüngliche Nachricht-
Von: t...@em.uni-frankfurt.de  
Gesendet: Donnerstag, 7. November 2019 07:58
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de   



Hi Herman,

Thanks for the suggestion. In principle, I have refined the structure without 
Arg and ligand, as well as the connected residues to the Arg.  
However, I still see the connected density of Arg and the ligand (Figure - 
unmodel1.png - only removal of Arg and ligand).

I have tried to refine the degraded product but the positive density in between 
Arg and the ligand is still there, indicating something should be modeled there 
(Figure - degraded_product.png and degraded_product2.png - both figures are the 
same ligand but with different side view).

I also tried to model the ligand in different conformation but still the same 
as shown in degraded product. The positive density is there between Arg and the 
ligand.

For both cases, I refined Arg with full occupancy but ligand with occupancy 
refinement. Now, Arg did not flip away from the density.

I might have a look at the mass-spec to check whether it is covalently linked 
although I am not aware of the reactivity of the moiety of the ligand and we 
never see this compound to covalent link with protein in our experience.

Thanks for the advice and suggestion. It is very helpful.

Thanks.

Best
HK



Quoting herman.schreu...@sanofi.com:

> Hi Heng-Keat,
>
> I had again a look at your electron density pictures and these were my 
> impressions:
> 1) the Arginine as fitted looks fully occupied. There are no hints for 
> an alternative conformations.
> 2) The fit of the ligand, although reasonable, is not extremely good.
>
> It seems that the only reason to invoke partial occupancies for the 
> Arginine and ligand are problems to fit both the ligand and the 
> arginine in the available density. This means that for the overlapping 
> parts, to sum of the occupancies is maximal 1.0.
> However, the ligand and the Arginine do not look half occupied.
>
> So I would look at other explanations:
> - would it be possible that the ligand reacts covalently with the 
> Arginine? The density looks pretty continuous to me.
> - could it be that instead of the intended ligand, some side- or 
> degradation product has bound?
>
> What I would also do, but what you probably already did, is to delete 
> the ligand and the Arginine side chain atoms and run several rounds of 
> refinement, to get an electron density map with as much bias as 
> possible removed.
>
> Good luck!
> Herman
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of 
> Heng-Keat Tam
> Sent: Mittwoch, 6. November 2019 07:30
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping 
> electron density of a residue and ligand
>
> EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk
>
>
>
> Hi Nestor,
>
> I think the reason for Arg side chain to curl up is because I refined 
> ligand and Arg side chain with occupancy refinement, and the Arg moved 
> away from the density, most likely due to 'repulsion' from ligand.
>
> The other question is: Is it possible that the H-bonds stay very 
> close? As I tried to 'real space refine' in coot, and the Arg side 
> chain flipped away from the density.
>
> Thanks for the suggestion.
>
> Best
> HK
>
>
> Quoting Nestor Concha :
>
>> Hi Tam,
>> The density looks very strong and therefore I'm going to guess that 
>> the Arg guanidimium stays in contact/interacts with the ligand and 
>> with the phosphate/sulfate next to it. Perhaps it is one of those 
>> close interactions with shorter H-bonds that usual given the 
>> arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a 
>> rotamer for the Arg that leaves the interactions intact rather than 
>> refine occupancies. Seems that the Arg side chain is curled up 
>> Nestor
>>
>> -Original Message-
>> From: CCP4 bulletin board  On Behalf Of 
>> Heng-Keat Tam
>> Sent: Tuesday, November 5, 2019 10:55 AM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron 
>> density of a residue and ligand
>>
>> EXTERNAL
>>
>> Dear Rob,
>>
>> I would like to model the alternative position for the side chain of
>> R120 but I don't really know whether the alternative 

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Hi Heng-Keat,

I had again a look at your electron density pictures and these were my 
impressions:
1) the Arginine as fitted looks fully occupied. There are no hints for an 
alternative conformations. 
2) The fit of the ligand, although reasonable, is not extremely good.

It seems that the only reason to invoke partial occupancies for the Arginine 
and ligand are problems to fit both the ligand and the arginine in the 
available density. This means that for the overlapping parts, to sum of the 
occupancies is maximal 1.0. However, the ligand and the Arginine do not look 
half occupied.

So I would look at other explanations: 
- would it be possible that the ligand reacts covalently with the Arginine? The 
density looks pretty continuous to me.
- could it be that instead of the intended ligand, some side- or degradation 
product has bound?

What I would also do, but what you probably already did, is to delete the 
ligand and the Arginine side chain atoms and run several rounds of refinement, 
to get an electron density map with as much bias as possible removed.

Good luck!
Herman

-Original Message-
From: CCP4 bulletin board  On Behalf Of Heng-Keat Tam
Sent: Mittwoch, 6. November 2019 07:30
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

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



Hi Nestor,

I think the reason for Arg side chain to curl up is because I refined ligand 
and Arg side chain with occupancy refinement, and the Arg moved away from the 
density, most likely due to 'repulsion' from ligand.

The other question is: Is it possible that the H-bonds stay very close? As I 
tried to 'real space refine' in coot, and the Arg side chain flipped away from 
the density.

Thanks for the suggestion.

Best
HK


Quoting Nestor Concha :

> Hi Tam,
> The density looks very strong and therefore I'm going to guess that 
> the Arg guanidimium stays in contact/interacts with the ligand and 
> with the phosphate/sulfate next to it. Perhaps it is one of those 
> close interactions with shorter H-bonds that usual given the 
> arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a 
> rotamer for the Arg that leaves the interactions intact rather than 
> refine occupancies. Seems that the Arg side chain is curled up 
> Nestor
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of 
> Heng-Keat Tam
> Sent: Tuesday, November 5, 2019 10:55 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron 
> density of a residue and ligand
>
> EXTERNAL
>
> Dear Rob,
>
> I would like to model the alternative position for the side chain of
> R120 but I don't really know whether the alternative conformation 
> exist as shown in the attached figure - density without the ligand and
> R120 overlaid with the refined structures of modeled ligand and R120.
> The ligand was modeled in two different conformations. From the 
> density, it seems to me that the density is connected or overlapped.
>
> It should not be a post-translation modification as it is well-known 
> that there is no post-translation modification for this protein.
> Furthermore, the crystal was obtained by co-crystallization of protein 
> with the ligand itself. The density seems to be the expected ligand.
>
> Thanks for the advice.
>
> Best regards
> HK
>
>
> Quoting Robert Nicholls :
>
>> Dear HK,
>>
>> No that's not quite correct - 'occupancy group alts complete' means 
>> that both R120 and the ligand are constrained so that their 
>> occupancies sum to unity. In contrast, 'occupancy group alts 
>> incomplete' means that the occupancies of R120 and the ligand will 
>> not be constrained to sum to unity (but the sum of their occupancies 
>> must be less than one). In both cases, R120 and the ligand will "see" 
>> each other in a certain sense. But, because they are assigned to 
>> different groups, it is assumed that they are present in different 
>> parts of the crystal. This means that they can overlap.
>>
>> Assuming that the ligand is in the correct conformation, I suspect 
>> the source of your problem is that you are modelling the side chain 
>> of
>> R120 as only one conformation. And I would also include the other 
>> atoms in the side chain - CB and CG.
>>
>> If you are modelling the sidechain of R120 with partial occupancies, 
>> then you should model those side chain atoms in two alternative 
>> positions (i.e. representing the portions of the crystal that 
>> do/don't have the ligand bound). This will help to ensure that your 
>> model makes physical sense. So the ligand plus the alt of R120 in the 
>> portion of the crystal that contains the ligand would be assigned to 
>> one occupancy group, and the alt of R120 in the portion of the 
>> crystal that does not contain the ligand would be assigned to the 
>> second group. In this case it would be appropriate to specify 
>> 

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Figure of merit in refinement

[Herman . Schreuder]

Hi James,

Did you ever try what happens if you set all the FOMs of e.g. 2vb1 to 1.0 and 
calculate a map? If you are sure they are not measurement errors they should be 
included in the map. I would expect some huge ripples, but maybe the "outliers" 
compensate and you get a truly interesting map.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von James Holton
Gesendet: Mittwoch, 16. Oktober 2019 17:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Figure of merit in refinement


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


All very true Randy,

But nevertheless every hkl has an FOM assigned to it, and that is used to 
calculate the map.  Statistical distribution or not, the trend is that hkls 
with big amplitude differences get smaller FOMs, so that means large 
model-to-data discrepancies are down-weighted.  I wonder sometimes at what 
point this becomes a self-fulfilling prophecy?  If you look in detail and the 
Fo-Fc differences in pretty much any refined structure in the PDB you will find 
huge outliers.  Some are hundreds of sigmas, and they can go in either 
direction.

Take for example reflection -5,2,2 in the highest-resolution lysozyme structure 
in the PDB: 2vb1.  Iobs(-5,2,2) was recorded as 145.83 ± 3.62 (at 5.4 Ang) with 
Fcalc^2(-5,2,2) = 7264.  A 2000-sigma outlier!  What are the odds?   On the 
other hand, Iobs(4,-6,2) = 1611.21 ± 30.67 vs Fcalc^2(4,-6,2) = 73, which is in 
the opposite direction.  One can always suppose "experimental errors", but ZD 
sent me these images and I have looked at all the spots involved in these hkls. 
 I don't see anything wrong with any of them.  The average multiplicity of this 
data set was 7.1 and involved 3 different kappa angles, so I don't think these 
are "zingers" or other weird measurement problems.

I'm not just picking on 2vb1 here.  EVERY PDB entry has this problem.  Not sure 
where it comes from, but the FOM assigned to these huge differences is always 
small, so whatever is causing them won't show up in an FOM-weighted map.

Is there a way to "change up" the statistical distribution that assigns FOMs to 
hkls?  Or are we stuck with this systematic error?

-James Holton
MAD Scientist
On 10/4/2019 9:31 AM, Randy Read wrote:
Hi James,

I'm sure you realise this, but it's important for other readers to remember 
that the FOM is a statistical quantity: we have a probability distribution for 
the true phase, we pick one phase (the "centroid" phase that should minimise 
the RMS error in the density map), and then the FOM is the expected value of 
the phase error, obtained by taking the cosines of all possible phase 
differences and weighting by the probability of that phase difference.  Because 
it's a statistical quantity from a random distribution, you really can't expect 
this to agree reflection by reflection!  It's a good start to see that the 
overall values are good, but if you want to look more closely you have to look 
a groups of reflections, e.g. bins of resolution, bins of observed amplitude, 
bins of calculated amplitude.  However, each bin has to have enough members 
that the average will generally be close to the expected value.

Best wishes,

Randy Read


On 4 Oct 2019, at 16:38, James Holton 
mailto:jmhol...@lbl.gov>> wrote:

I've done a few little experiments over the years using simulated data where I 
know the "correct" phase, trying to see just how accurate FOMs are.  What I 
have found in general is that overall FOM values are fairly well correlated to 
overall phase error, but if you go reflection-by-reflection they are terrible.  
I suppose this is because FOM estimates are rooted in amplitudes.  Good 
agreement in amplitude gives you more confidence in the model (and therefore 
the phases), but if your R factor is 55% then your phases probably aren't very 
good either.  However, if you look at any given h,k,l those assumptions become 
less and less applicable.  Still, it's the only thing we've got.

2qwAt the end of the day, the phase you get out of a refinement program is the 
phase of the model.  All those fancy "FWT" coefficients with "m" and "D" or 
"FOM" weights are modifications to the amplitudes, not the phases.  The phases 
in your 2mFo-DFc map are identical to those of just an Fc map.  Seriously, have 
a look!  Sometimes you will get a 180 flip to keep the sign of the amplitude 
positive, but that's it.  Nevertheless, the electron density of a 2mFo-DFc map 
is closer to the "correct" electron density than any other map.  This is quite 
remarkable considering that the "phase error" is the same.

This realization is what led my colleagues and I to forget about "phase error" 
and start looking at the error in the electron density itself 
(10.1073/pnas.1302823110).  We did this rather pedagogically.  Basically, 
pretend you did the whole experiment again, but "change up" the source of error 
of interest.  For example if you want to see the effect of 

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] no tNCS and Phaser manuals

[Herman . Schreuder]

Dear Iracema,

Thank you very much for your hint of using CCP4i, I never use it but this time 
I should have used it to find out how to switch off the tNCS. Unfortunately, I 
cannot reach any of the phaser.cimr sites. I get the error message that our 
proxy server does not react. I can reach the phaser wiki at slac, but that one 
seems to be most recently updated in 2016 and most of the links produce “page 
not found” error messages.

I probably should contact our IT department what is wrong with our proxy 
server, since when our firewall blocks a site, it will tell so, which is not 
the case here.

Best,
Herman


Von: IRACEMA CABALLERO MUÑOZ 
Gesendet: Dienstag, 1. Oktober 2019 15:06
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] no tNCS and Phaser manuals


EXTERNAL : Real sender is icm...@ibmb.csic.es

Hi Herman, I made an image that shows how to turn off tNCS in CCP4i, CCP4i2 and 
Phenix.
[phaser_tncs.jpg]

In addition in the phaser wiki there are several tutorials and some other 
useful information:
https://www.phaser.cimr.cam.ac.uk/index.php/Tutorials


Best wishes,
Iracema Caballero

El mar., 1 oct. 2019 a las 14:45, 
mailto:herman.schreu...@sanofi.com>> escribió:
Dear community,

I wanted to switch off the tNCS for a Phaser run, but could not find any Phaser 
manuals online, explaining how to do this.
So I have two questions:

  1.  How do I switch off tNCS in Phaser and
  2.  Are there any phaser manuals online? I tried hard to find them using 
Bing, but the websites were either not present, not available or could not be 
found. It may be that our firewall is also blocking certain sites although most 
sites are accessible.

Thank you very much for your help!
Herman






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[ccp4bb] no tNCS and Phaser manuals

[Herman . Schreuder]

Dear community,

I wanted to switch off the tNCS for a Phaser run, but could not find any Phaser 
manuals online, explaining how to do this.
So I have two questions:

  1.  How do I switch off tNCS in Phaser and
  2.  Are there any phaser manuals online? I tried hard to find them using 
Bing, but the websites were either not present, not available or could not be 
found. It may be that our firewall is also blocking certain sites although most 
sites are accessible.

Thank you very much for your help!
Herman






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[ccp4bb] AW: [EXTERNAL] [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Herman . Schreuder]

Dear Chris,

The first thing I would do is to load the FFT map (not the mtz) in coot and see 
how densities of both ligands compare in that case. Also in FFT, did you 
calculate exactly one unit cell, or did you select a region just around the 
protein? If you contour the maps in terms of RMSD, the amount of solvent 
included in the maps influences the RMSD level. For comparison it would be 
better to look at contour levels in electrons/Å**3. Finally, you could also 
look at the contour levels of the surrounding protein atoms, to get an idea how 
the ligand density compares to the protein density.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Chris Fage
Gesendet: Montag, 30. September 2019 12:37
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol


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

Dear All,

I recently obtained structures of a protein bound to two different small 
molecules. When viewing the structures in Coot with a similar contour setting, 
the 2Fo-Fc map around ligand 1 is clearly much weaker than that around ligand 
2.However, after generating 2Fo-Fc maps in FFT and loading them in Pymol 
(again, choosing equal contour levels), the maps surrounding ligands 1 and 2 
have nearly the same quality. Is there a difference in scaling between the two 
programs that can account for this? Thanks for any advice!

Best wishes,
Chris

[https://ipmcdn.avast.com/images/icons/icon-envelope-tick-green-avg-v1.png]

Virus-free. 
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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] AW: [ccp4bb] challenges in structural biology

[Herman . Schreuder]

Dear John,
Plants cannot walk away to a more favorable spot. They remain stuck where they 
germinate, e.g. whether the place is sunny, shady, wet, dry, fertile, poor etc. 
So plants compensate by having a lot of genes available to be able to adapt to 
the particular spot where happen to be. And indeed, plants have usually more 
genes then animals!
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von John R 
Helliwell
Gesendet: Freitag, 20. September 2019 09:19
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: [ccp4bb] challenges in structural biology


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

Dear Martin,
Many thanks for these details of the size of the human genome over the decades 
and also the news of your most interesting upcoming review. I shall read it 
with great interest.
Incidentally is the over 4 genes for the rice genome number correct? This 
number caught my eye as being interesting how the rice genome is more 
complicated than our genome.
Best wishes,
John
Emeritus Professor John R Helliwell DSc




On 19 Sep 2019, at 08:35, Kollmar, Martin 
mailto:m...@nmr.mpibpc.mpg.de>> wrote:
Dear John,
the „100,000 human genes“ is a long-standing myth broad forward by the 
initiators of the U.S. human genome sequencing projects in 1990. This large 
number completely contradicted all genetics and mutation data since the 1940th, 
but the sequencing community (genome, cDNA, EST) didn’t read even the standard 
text books. Thus, the “30,000” genes published with the two human genome papers 
in 2001 are not “surprisingly low” but just in accordance with the predictions 
and the data since the 1940th. The gene number went down to about 23,000 
already in 2004, and the current numbers (depending on database) range around 
20,000 human protein-coding genes. The myth of the large numbers is only 
propagated by those who profit from larger numbers (e.g. bigger grants, papers 
in higher IF journals, big consortia).

I have written a review about the current state (and history) of the human 
protein-coding genes, which will appear online in BioEssays soon and finally in 
the November issue (will be open access). In this review there will be some 
(hopefully) useful plots showing the gene numbers since the 1940th and a 
detailed review of all the numbers and their experimental basis (most were 
actually just extrapolations from small-scale data).

Please excuse this kind of self-advertisement, but it is really more than time 
to move this myth out of science literature and communication.

Best regards,
Martin

Priv. Doz. Dr. Martin Kollmar

Group Systems Biology of Motor Proteins
Department NMR-based Structural Biology
Max-Planck-Institute for Biophysical Chemistry
Am Fassberg 11
37077 Goettingen
Deutschland

www.motorprotein.de
 (Homepage)
www.cymobase.org
 (Database of Cytoskeletal and Motor Proteins)
www.diark.org
 (diArk - a resource for eukaryotic genome research)
www.webscipio.org
 (Scipio - eukaryotic gene identification)

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von John R Helliwell
Gesendet: Donnerstag, 19. September 2019 08:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] challenges in structural biology

Dear James,
Well, 100,000 genes used to be the estimate of the size of the human genome.
(eg see 
https://physicsworld.com/a/protein-crystallography-the-human-genome-in-3-d/
 )
It seems it has got easier, albeit still gargantuan, at ~30,000 genes to be 
expressed into proteins.

Meanwhile funding agencies also look out for Big Ideas:-

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] not solely pdb issue: need someone to officially settle the pdb dispute

[Herman . Schreuder]

Being denied a job because someone else published your research may make the 
dance also somewhat less happy…

Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark J 
van Raaij
Gesendet: Mittwoch, 21. August 2019 11:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] not solely pdb issue: need someone to 
officially settle the pdb dispute


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

Another problem is that the structure was apparently originally not cited 
properly, and now still cited as work from the lab of B, rather than as work of 
A...

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616

On 21 Aug 2019, at 11:22, Anastassis Perrakis 
mailto:a.perra...@nki.nl>> wrote:

If the structure has been deposited in the PDB and thus is public ally 
available, B (or F, G, Φ, Ξ, Δ, Α or whoever else) has every right to use it in 
a publication.

“A” should follow the advice of Frank and do a happy dance for the usefulness 
of the work, or if not feeling like dancing she/he could follow my advice that 
will be offered in Greek: «ξυδάκι».
Sent from my iPhone

On 21 Aug 2019, at 11:13, Flemming Goery 
mailto:flemming_go...@hotmail.com>> wrote:
Dear all:
A has sought a job in the lab of B. B invited A for a interview with a PPT oral 
presentation, as requested B has sent the PPT on the structural biology 
research of XXX to B by e-mail, and presented in front of A and his 
postdoctoral researcher.

After interview, B requested all research documents (including detailed 
reports) on XXX to be sent by A to B by e-mail, A sent, including 2 sets of pdb 
for the same structure, one set with solvent, one without. A told B all 
intellectual property of the Documents and the research belonged to A, based on 
the regulation of A's institute.

B sought a referee from A's institute, to someone A did not agree. It seems the 
referee told B one set of PDB has been deposited (the one without solvent)

Then B did not give the offer to A. A joined Institute D, without independent 
funding for the writing (in fact, no salary to support this writing, and no fee 
for publication of this work).

Several years later, A found B's paper, i.e., the concerned paper published in 
Journal C. In the paper, B has used the information from deposited PDB for 9 
times (already a significant paprt of the paper, not to say the message from 
the other Documents sent to B by A). In the paper, it write something like, 
'based on our work on the structure of  (folowed by 4 letter pdb code)', which 
implied the structure was solved by the authors of the paper, rather than by A.

A contacted Journal C, Journal C contacted B, B claimed the deposited PDB was a 
public domain knowldge. Journal C took the action to add the reference to the 
deposited pdb in the paper.

As mentioned, the paper has mentioned and used the message from the deposited 
pdb 9 times, and in the paper the reference mark was not added to the first 
occurence of the mentioning of the deposited pdb, but added (only once for the 
9 occurences of depositation code) to a paragraph where it can be concluded 
that the authors have used the undeposited pdb with the solvent. In another 
words, although reference to the deposited pdb was added by a correction, from 
where the reference mark was added, it cannot show they have refered to the 
cited pdb, not to say the undeposited pdb with solvent which they used based on 
the paragraph information.

A's concern was that: A cannot exclude the possibility that the research in the 
paper other the part related to PDB, were fabricated, thus A request paper 
retraction as the major clain.

If cannot retratcted, A request to be the correspondence author (sometimes 
requets co-first author, sometimes request both co-first author and 
co-correspondence author), as without A's work (the PPT presentation, 2 sets of 
pdb, all documents), the work in the concerned paper cannot be done. A regard 
as having contributed to the initiation of the paper, thus A prefer to be add 
as a co-correspondence author if appropriate.

First, can the paper deserve a retraction, and second, can B deserve a 
co-author?

Flemming



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[ccp4bb] AW: need someone officially settle a pdb dispute for a publication

[Herman . Schreuder]

Dear Flemming,

As Jürgen said, what happened? Did A deposit the coordinate file in the pdb, 
but did not publish and did B take this coordinates and make a publication? Or 
did B ask A for the coordinates to have a look at and then made a publication 
without agreement of A? Did B hack the computer account of A and stole the 
coordinates?

In general, if a significant part of the publication of B were based on the 
coordinates produced by A, A should be coauthor. However, if the coordinates 
were deposited and A had ample time to write a publication, but did not do so, 
as Jürgen said, all A could ask for is to have his structure properly cited and 
acknowledged.

Something about tactics: Institute heads tend to protect their own people, so 
if the head of institute B gets an email from someone he/she does not know 
complaining about one of the people of his/hers institute, the institute head 
may not find the time to go through the trouble of a misconduct investigation 
with may harm the reputation of the institute.

However, if the head of institute A would learn that one of its people has been 
plagiarized by someone of institute B, the motivation to react will be much 
higher, especially if institute A and B are in a friendly competition.

So if I were A, I would go the head of institute A and complain about the 
academic misconduct of B and discuss what steps could be taken.

My 2 cents,
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Flemming 
Goery
Gesendet: Dienstag, 20. August 2019 17:46
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] need someone officially settle a pdb dispute for a 
publication


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

Dear All,

A and B belong to 2 different institutes. A claimed B has used his pdb for a 
publication in Journal C. Journal C did not give the retraction, but permit 
complain related to the journal publication author issue, with the prerequisite 
journal C did not have the authority on authorship dispute. Then A has e-mailed 
to the institute head of B with academic misconduct by B as claim, the 
institute head of B did not give reply.

In this situation, can A have the journal  authorship  dispute settled by a 
neutral reviewer (Journal C view: you (A) need to reach out to the institutions 
that have authority to adjudicate on such matters, as investigation and 
adjudication on authorship claims falls outside the remit of journal editors. 
)? Who are qualified as the neutral reviewer so that the review decision can be 
submitted to Journal C?

If you believe you are qualified, or you know somebody or some organization 
qualified, please let me know and I will introduce the issue to you by separate 
e-mail (it is best not disseminated, am I right?)

Best regards.

Flemming




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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Extra density close to phosphate bound to Zn2+

[Herman . Schreuder]

PS, you also have to define an alternative conformation, but that you probably 
already did.

Von: Schreuder, Herman /DE
Gesendet: Montag, 5. August 2019 17:55
An: 'Maria Håkansson'; CCP4BB@JISCMAIL.AC.UK
Betreff: AW: [EXTERNAL] Re: [ccp4bb] Extra density close to phosphate bound to 
Zn2+

Hi Maria,

Did you rotate the phosphate, or invert it? If you invert the phosphate, you 
may get into trouble with the parameters. Although a phosphate is symmetric, 
its oxygens have different names and inverting it leads to all kind of 
problems, especially in a high resolution map which does not allow the 
phosphate to flip it back in an “allowed” conformation.

If you did rotate the phosphate, I take my words back and you may have to look 
for ions.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Maria 
Håkansson
Gesendet: Montag, 5. August 2019 17:32
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Extra density close to phosphate bound to Zn2+


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

Hi,

Thanks for your suggestions but I have tried to invert the phosphate and it is 
not
fitting the map. The geometry is not correct that way and it is too good data 
to ignore and
to my knowledge a pentacoordinated phosphate is a non existent species.

That leaves me with ions.

Best regards,
Maria


Maria Håkansson, PhD, Crystallization Facility Manager
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com




On 5 Aug 2019, at 16:32, Jan Abendroth 
mailto:jan.abendr...@gmail.com>> wrote:

Hi Maria,
apart from the suggestions that were already made, take a look at the P-O bond 
on the right side of the P. Maybe it is just the perspective, this appears to 
be rather long.
So, instead of the alternate conformation, maybe just a flip of the phosphate 
and a water off to the right?

Cheers,
Jan

On Mon, Aug 5, 2019 at 7:16 AM Nukri Sanishvili 
mailto:sannu...@gmail.com>> wrote:
Hi Maria,
Let's not ignore the "missing" density - the red one. If the question is only P 
vs S, a sulfur would add to the negative density, i.e. make matters worse. It 
also appears that modeling a phosphate in alternative conformations, as 
suggested by Wim and Roger, would take care of the issue of negative density as 
well.
Cheers,
Nukri

On Mon, Aug 5, 2019 at 8:05 AM Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:
Dear CCP4 bulletin board,

I am working with some lytic enzymes called endolysines, which
bind Zn2+ in the active site. I have three homologues protein
determined to 1.2 Å each where the Zn2+ is bound
to a cystein, two histidines and one phosphate ion added (1.9-2.3 Å binding 
distances) in the crystallization experiment.

Now to my question. Close to the phosphate (B-factor=20Å2) ion a 8 sigma peak 
is present in all three endolysines, see below.
I have modeled it to a Na+ (B-factor= 30 Å2) or a Li+ (B factor = 13Å2) ion.
Sodium has benn added in the crystallization experiments since sodium potassium 
phosphate
salt has been used. The only reason for including Li+ is that I think the 
binding distances (1.7-2.0 Å) are too short for Na+.

I have also tried to make a model with the phosphate in two different 
conformations but it does not fit.

Have anyone seen something similar before? What is the most correct way of 
dealing with unknown densities?
It is difficult to disregard +8 sigma difference density close to the active 
site.

Thanks in advance for any help!

Best regards,
Maria

 

Maria Håkansson, PhD, Crystallization Facility Manager
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com






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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Extra density close to phosphate bound to Zn2+

[Herman . Schreuder]

Hi Maria,

Did you rotate the phosphate, or invert it? If you invert the phosphate, you 
may get into trouble with the parameters. Although a phosphate is symmetric, 
its oxygens have different names and inverting it leads to all kind of 
problems, especially in a high resolution map which does not allow the 
phosphate to flip it back in an “allowed” conformation.

If you did rotate the phosphate, I take my words back and you may have to look 
for ions.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Maria 
Håkansson
Gesendet: Montag, 5. August 2019 17:32
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Extra density close to phosphate bound to Zn2+


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

Hi,

Thanks for your suggestions but I have tried to invert the phosphate and it is 
not
fitting the map. The geometry is not correct that way and it is too good data 
to ignore and
to my knowledge a pentacoordinated phosphate is a non existent species.

That leaves me with ions.

Best regards,
Maria


Maria Håkansson, PhD, Crystallization Facility Manager
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com





On 5 Aug 2019, at 16:32, Jan Abendroth 
mailto:jan.abendr...@gmail.com>> wrote:

Hi Maria,
apart from the suggestions that were already made, take a look at the P-O bond 
on the right side of the P. Maybe it is just the perspective, this appears to 
be rather long.
So, instead of the alternate conformation, maybe just a flip of the phosphate 
and a water off to the right?

Cheers,
Jan

On Mon, Aug 5, 2019 at 7:16 AM Nukri Sanishvili 
mailto:sannu...@gmail.com>> wrote:
Hi Maria,
Let's not ignore the "missing" density - the red one. If the question is only P 
vs S, a sulfur would add to the negative density, i.e. make matters worse. It 
also appears that modeling a phosphate in alternative conformations, as 
suggested by Wim and Roger, would take care of the issue of negative density as 
well.
Cheers,
Nukri

On Mon, Aug 5, 2019 at 8:05 AM Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:
Dear CCP4 bulletin board,

I am working with some lytic enzymes called endolysines, which
bind Zn2+ in the active site. I have three homologues protein
determined to 1.2 Å each where the Zn2+ is bound
to a cystein, two histidines and one phosphate ion added (1.9-2.3 Å binding 
distances) in the crystallization experiment.

Now to my question. Close to the phosphate (B-factor=20Å2) ion a 8 sigma peak 
is present in all three endolysines, see below.
I have modeled it to a Na+ (B-factor= 30 Å2) or a Li+ (B factor = 13Å2) ion.
Sodium has benn added in the crystallization experiments since sodium potassium 
phosphate
salt has been used. The only reason for including Li+ is that I think the 
binding distances (1.7-2.0 Å) are too short for Na+.

I have also tried to make a model with the phosphate in two different 
conformations but it does not fit.

Have anyone seen something similar before? What is the most correct way of 
dealing with unknown densities?
It is difficult to disregard +8 sigma difference density close to the active 
site.

Thanks in advance for any help!

Best regards,
Maria

 

Maria Håkansson, PhD, Crystallization Facility Manager
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: 
www.saromics.com






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--
Jan Abendroth
Emerald Biostructures
Seattle / Bainbridge Island, WA, USA
home: 

[ccp4bb] AW: Bond between Cys S and Met methyl group?

[Herman . Schreuder]

Hi Stephen,

What happens if you delete the CE methyl group and run a round of refinement. 
Do you get a positive blob of difference density in between the two sulfurs? Or 
looks everything pretty ok? In the first case you may indeed have some unusual 
phenomenon, in the latter case, the methyl group is probably not linked to the 
cysteine, but at some disordered, invisible position away from the cysteine. 
This would be the more usual (and boring) explanation.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Stephen 
Graham
Gesendet: Mittwoch, 17. Juli 2019 16:03
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Bond between Cys S and Met methyl group?

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



Hi all,

We've spotted something very weird in our density that we're struggling to 
reconcile...we have two sulfur atoms (from Met and Cys residues) that are very 
close together. The density looks for all the world like the methyl group from 
the Met should also be bonding to the Cys, although obviously the non-bonded 
terms are keeping them apart during refinement. The dmin is 1.72 and R/RFree 
are ~0.18/0.20 so the density should be pretty believable. We've seen the same 
in two crystals. Interestingly, we don't see much change in density if we 
process just the first/last quarter of the dataset so it isn't overly sensitive 
to radiation damage during collection.

I posted a short vid on Twitter to illustrate: 
https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_i_status_1151488524425814016=DwIFAw=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=AGJpHgNJ7OJQ60bcbZONEofZkhRwnQqnDQtlX6xRQVY=10R2PgUMqqcCQ2fADadiNWCxz-zY9dt4V8mJt_oPSFE=

Has anyone ever seen something like this? Is a 
(Cys)CA-CB-SG-CE-SD-CG-CB-CA(Met) bond possible?

Thanks,

Stephen

--
Dr Stephen Graham
Sir Henry Dale Fellow and University Lecturer
Department of Pathology
University of Cambridge
Tennis Court Road
Cambridge CB2 1QP
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.path.cam.ac.uk_research_investigators_graham_=DwIFAw=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=AGJpHgNJ7OJQ60bcbZONEofZkhRwnQqnDQtlX6xRQVY=1Wxlqn5ABBEZSTuhDIUatEkDPjr78DB_M6NHrD6xsyY=



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] twinning problems

[Herman . Schreuder]

Dear Marina,

Just an observation from my side: if you are able to process your data in P622, 
there must be some 2-fold perpendicular to l, which would most likely be a 
non-crystallographic or a twinning axis. Is there a NCS 2-fold in the P321 data 
set you solved? If so, is it perpendicular to l? If not, there must be some 
twinning law that converts l into -l like -k,-h,-l

Also, careful analysis of your solved P321 structure could give some hints how 
the twinning might look like.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Marina 
Ley
Gesendet: Donnerstag, 11. Juli 2019 12:50
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] twinning problems

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



Hello everyone,

I have the following problem:

I have some really good data sets of about 1.6 A which could be  
processed in P 6 2 2 as well as in P 3 2 1.
Intensity statistics tests indicate twinning (merohedral, twinning  
law: -h, -k, l, in P 3 2 1).
But I did not find a good MR solution (R value around 60%) and when I  
try to refine it with the twin law I get really bad maps.

However I could get one data set where the intensity statistics tests  
did not indicate twinning and I have already managed to solve the  
structure.

SPACE_GROUP_NUMBER:  154 (P 3 (2) 2 1)
UNIT_CELL_CONSTANTS:57.5657.56   159.98  90.000  90.000 120.000
number of molecules in asymmetric unit: 2
NCS: yes

I could see a lot of amino acids in the electron density which were  
not in the search model.

But it seems like most of my crystals are twinned. For a fragment  
screen which I would like to do I need to solve the twinned data sets,  
otherwise I have to search again for another crystallization condition.

I don't know what to do next to solve the twinned data sets. Do you  
have any ideas or suggestions?

Thanks in advance!!!

Cheers,
Marina



Some information about the results from Xtriage:

 --Statistics independent of twin laws--

   /^2 : 1.586  (untwinned: 2.0, perfect twin: 1.5)
   ^2/ : 0.870  (untwinned: 0.785, perfect twin: 0.885)
   <|E^2-1|>   : 0.564  (untwinned: 0.736, perfect twin: 0.541)
   <|L|>   : 0.380  (untwinned: 0.500; perfect twin: 0.375)
  : 0.208  (untwinned: 0.333; perfect twin: 0.200)
   Multivariate Z score L-test: 12.491



-- 
Marina Ley

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

https://urldefense.proofpoint.com/v2/url?u=http-3A__www.agklebe.de_=DwIBaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=O01KIRRRZ9Krf6AS9sPy8bgILSyqYf592SmrmmH_H6I=OiBXd3CAqkfdaGH-2fS0EaH-dsY6ThKMKfqCYqiskZE=



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] tNCS incompatible with cell dimensions

[Herman . Schreuder]

Dear Kevin,

It could also be that you have a particular nasty combination of tNCS and 
twinning. Given your packing problems in the ab plane, this would mean that 
your 2-fold parallel to c is generated by twinning and that probably one of the 
21 axes is generated by twinning as well.

With some very clever thinking, you might be able to figure out what the 
appropriate lower-symmetry space group would be, but I would follow the advice 
of John Helliwell: reprocess the data in P1 and run molecular replacement in 
P1. MR is usually quite insensitive to twinning and will produce two solutions 
with equal probability. Once you have the packing, you can try to figure out 
how to best describe this packing in terms of a space group with tNCS. If your 
data collection statistics do not suggest any twinning, you may have some other 
pathology like statistical disorder.

In any case, with this space group problem, you have great opportunity to learn 
a lot about crystallography!

Best,
Herman




Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Kevin 
Jude
Gesendet: Freitag, 31. Mai 2019 22:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] tNCS incompatible with cell dimensions


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

Hello community, I wonder if I could solicit advice about a problematic 
dataset. I plan to solve the structure by molecular replacement and expect that 
the protein is relatively compact, ie not elongated. SAXS data supports this 
expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a 
= 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
vector of {0.5, 0.5, 0}.

If you're sharper than me, you may have already spotted the problem - c is the 
long axis of the unit cell, but tNCS constrains the proteins to a plane 
parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser 
will not give a solution in any orthorhombic space group unless I turn off 
packing, and then I get large overlaps in the a,b plane and huge gaps along c.

Since I believe that my model is good (or at least the correct shape, based on 
SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights 
into how to approach this problem would be much appreciated.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Disulphide occupancies.

[Herman . Schreuder]

Dear Jonathan,

In these cases, I usually see positive difference density nearby, indicating an 
alternative position for one of the sulfurs, i.e. the disulfide bridge was 
partly broken. I am too lazy to fit these, but if you want to do a perfect job, 
you might want to fit an alternative conformation for this sulfur. You may have 
to create an alternative conformation for the other sulfur as well to have one 
bonded disulfide bridge and one open one.

Considering deposition: you are the depositor. If you believe that the model 
you made is the most faithful representation of the “true” structure, you 
should deposit it and the pdb will have to accept. In the worst case they will 
add some warnings.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jonathan 
Cooper
Gesendet: Montag, 27. Mai 2019 00:20
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Disulphide occupancies.


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

When you refine structures with disulphide bridges you often get negative 
difference density for the sulphurs, presumably due to the well-known radiation 
damage effects. The negative difference density often won't disappear with 
usual B-factor refinement. However, it seems to go away if you refine the 
occupancy of the affected sulphur atoms e.g. to 0.9 or thereabouts. Would it be 
acceptable to publish/deposit structures where the sulphur occupancy is less 
than one, given a suitable REMARK in the pdb file? Thank you.



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] crystals mounted in microRT capillaries

[Herman . Schreuder]

For glass X-ray capillaries, we used a Styrofoam box with some cooling blocks 
at room temperature to prevent any fast temperature changes, which may lead to 
condensation problems. Of course, you should add some materials to protect the 
capillaries and keep them in place.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Michael 
Colaneri
Gesendet: Freitag, 17. Mai 2019 08:20
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] crystals mounted in microRT capillaries


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

Dear all,

how can one mail crystals mounted in loops using the microRT capillaries of 
Mitegen?

Thank you.

Mike Colaneri



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring

[Herman . Schreuder]

Dear Sam,

I would remove the ice ring and reprocess the data. Ice rings may wreak havoc 
with scaling so at minimum you have to redo the scaling.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Sam Tang
Gesendet: Donnerstag, 4. April 2019 11:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] High Rfree - ice ring


Dear Eleanor and Eric

Thanks for your replies.

Yes indeed when we looked at the plots e.g. R factor vs resln there was a sharp 
peak near 3.6 - 3.8 A which is where we visibly saw an ice ring on the image. 
Thus our first thought was to remove the ic ring. (either reprocess or can we 
bypass this resolution range during refinement?)

The protein is 50 kDa, two molecule in the ASU, seemingly no obvious density 
was unassigned. We got ~3 total observations, ~15000 unique observations. 
NCS restraints was applied.

Best regards
Sam



On Thu, 4 Apr 2019 at 08:57, Eric Montemayor 
mailto:montemayor.e...@gmail.com>> wrote:
That’s a rather large gap between Rwork and Rfree.  I suspect you have 
mis-assigned your space group and as a result have a large number of copies in 
your asymmetric unit.  Any structure can be solved in P1, but that does not 
mean the true space group is indeed P1. If you use P1 when it’s not actually 
P1, you will have an unnecessarily overparamerized model, hence the large gap 
between Rwork and Rfree.

Questions:
1- how many copies in your asymmetric unit in P1?
2- how many atoms in your model vs number of unique reflections?
3- if more than one copy per asymmetric unit, are you imposing NCS restraints 
during refinement?

-Eric



On Wed, Apr 3, 2019 at 1:41 PM Sam Tang 
mailto:samtys0...@gmail.com>> wrote:
Hi everyone again

Hmmm I think we have solved a structure in P1 space, to 2.5 A. However after 
refinement the Rfree stuck at 33%-35% with Rwork around 26%. The structure was 
solved by MR and current model seems to fit density well. In Refmac log I found 
that at the resolution corresponding to high R there may be a solvent/ice ring. 
Since imosflm should be able to exclude ice rings, I am not 100% sure whether 
it's the cause to high R. But if this is actually the case, is there a way I 
can exclude certain resolution bins during Refmac (and is it an appropriate way 
to do so?)

PS - the data is not affected by twining or pseudosymmetry as checked by 
Xtriage.

Many thanks!

Sam



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] refmac5 problems with unnatural amino acid

[Herman . Schreuder]

Dear Deniz,

In the past, I had similar problems caused by the fact that when the residue 
was “known” to ccp4, it would use the cif file from the ccp4 library instead of 
the cif I had created myself. You could check if this might be the case for 
you. You find the cif files under $CCP4/lib/data/monomers in the subdirectory 
with the first letter of the three-letter code of your ligand and check the CIF 
file with the same three-letter code as you are using.
However, it is possible that the ccp4 developers have resolved this issue by 
letting an externally provided cif file override the internal cif file.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von "Deniz 
Üresin"
Gesendet: Mittwoch, 3. April 2019 13:15
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] refmac5 problems with unnatural amino acid

Hello,
I'm trying to refine the structure of a protein mutant that has an unnatural 
amino acid in it. I created a restraint file for the AA and the mutation worked 
fine in COOT. But when I use refmac5 for refinement, it always changes the bond 
angle in the sidechain (an azide group, which should have a bond angle of 
180°). I set the SD of the angle to 0 but that didn't help (the "refined" bond 
angle ends up somewhere between 120 and 150°).
Is there any other way to tell refmac5 to not alter that bond angle?
Thanks in advance,
Deniz Üresin

_
Deniz Üresin
Institut für Biophysik
Johann Wolfgang Goethe-Universität Frankfurt am Main
http://www.biophys.uni-frankfurt.de/~bredenbeck/




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[ccp4bb] AW: [ccp4bb] pseudo internal symmetry

[Herman . Schreuder]

I agree. The data processing software might have been confused by the NC 2-fold 
and thought that it was crystallographic and of course, after merging the data, 
the NC axis will have been made “crystallographic” with superimposed A and B 
conformations at 50% occupancy. In this case, reprocessing in the true, lower 
symmetry space group, would bring better stats, but also much clearer and much 
easier interpretable electron density maps. However, if the distribution of 
you’re A and B conformations is truly random you could submit the structure as 
it is now.
Best, Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von David 
Briggs
Gesendet: Mittwoch, 3. April 2019 10:46
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] pseudo internal symmetry

Hi,

This doesn't answer your question directly, but I'd be tempted to reduce the 
symmetry and re-refine in a space group lacking that 2-fold axis.
I'd hope that the scaling in the lower space group would give you better stats 
as well, but maybe the differences in your case are too subtle?

I don't know what PDB rules and regs are though.

HTH,

Dave

From: CCP4 bulletin board  on behalf of Daniele de 
Sanctis 
Sent: 03 April 2019 09:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo internal symmetry

Hi all,

we have a structure with a pseudo internal symmetry along a 2-fold axis that 
sits on a 2-fold crystallographic axis. For refinement purposes we have modeled 
the parts that differ with 50% occupancy, but before depositing the structure 
we wanted to make sure that this is the best way to deal with it and it is in 
agreement with PDB standards.

Did anyone deal with similar cases in the past?

Cheers

Daniele


--

ἀρετή
---
Daniele de Sanctis
Structural Biology Group
ESRF - The European Synchrotron
Grenoble, France
Tel 33 (0)4 76 88 2869

http://www.esrf.eu/id29



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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Refinement

[Herman . Schreuder]

Dear ???,
Do you have any ice rings (even hardly visible ones) in your diffraction data?
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von StrBio
Gesendet: Sonntag, 24. März 2019 05:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Refinement

ALL.

I have data at 2.4 A in P21 sp gr, helical protein.
Refined to Rwork 29 Rfree 34 with nice density map and all nice statistics 
oither Rfactor (by Phenix). Refmac quit same.
Should I deposit it or look better data?
Any suggestion?





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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] crystal contacts and biologically relevant contacts

[Herman . Schreuder]

Hi Alex,

Kevin is right. To clarify things a little, you don’t have to use the symmetry 
mate selected by the molrep program. You can take the symmetry mate that makes 
the relevant biological interactions instead. No need to change the space group.

You have to delete the molecule that does not make the relevant contacts and 
add the symmetry mate of this molecule that does make the relevant contacts and 
maybe run one more cycle of refinement to get rid of rounding errors. (and make 
sure that the procedure you used is correct)

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Kevin 
Jude
Gesendet: Freitag, 22. März 2019 04:11
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] crystal contacts and biologically relevant 
contacts

Hi Alex,
Yes this is acceptable - symmetry-related proteins are identical and 
interchangeable. It can be helpful to use the PISA program (a plugin for Coot 
or as a webserver 
http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver)
 to identify which symmetry-related proteins are forming the biological unit, 
as well as what you know from biochemical studies (not to mention the position 
of CDRH3, etc).

The easiest way to make the change, IMHO, is to open your molecule in Coot, 
center on the appropriate symmetry mate, and use Extensions>Modelling>Symm 
Shift Reference Chain Here.
--

Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431


On Thu, Mar 21, 2019 at 7:38 PM Liu Jingxian, Alex 
mailto:ljx...@gmail.com>> wrote:
Dear Colleagues,
Recently I solved one Ab-Ag complex structure. It was indexed as p212121 or 
p21212 space group and solved the structure with one Fab and one Ag in the cell 
unit. The contact interface is not the one I expected (elbow region contacts 
with Ag). Using symexp I got several symmetry related molecules, showing the 
interface of antibody CDR with the antigen.

For non-structural biologist it is quite tricky to display PDB file with 
symmetry related files. To make things simple, I want to solve the structure 
with the biologically relevant complex in one AU, However, changing space 
groups (such as P1 or P2) didn't help and I failed in the following steps. Some 
people really added the symmetry related molecules into the final pdb (merge 
and rename the chain ID of the mate molecules). So is it acceptable in 
structure community? Many thanks.

Best Regards,
Jingxian



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Change dimer assembly in ASU

[Herman . Schreuder]

Dear Ezequiel,

Be careful, it also happens that the asymmetric contains two half-dimers, with 
the other half of the dimers being generated by crystallographic operators.

In this case it is not possible to rearrange the monomers such that the 
asymmetric unit contains one biological dimer and for refinement, one has to 
stick to the arrangement obtained by molecular replacement. However, for 
analysis and the making of pictures, it is perfectly valid to generate the two 
biological dimers using the appropriate crystallographic symmetry operators. 
WITHIN the dimers generated that way, the monomers will be identical, however, 
BETWEEN the two dimers, there might be (small) differences, which may or may 
not be biologically relevant.

Good luck!
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Dienstag, 5. März 2019 13:04
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Change dimer assembly in ASU

I tend to use PISA for suggesting biologically relevant complexes.  And you can 
modify the MR solution to replace a molecule with  any symmetry generated copy 
of it..

But check Phaser has put your molecule reasonably close to the origin.



On Mon, 4 Mar 2019 at 19:52, Randy Read 
mailto:rj...@cam.ac.uk>> wrote:
Dear Ezequiel,

There is nothing special about which particular symmetry copies a molecular 
replacement program chooses, so there is no good reason to stick to those 
symmetry copies.  On the other hand, there are very good reasons to present a 
molecule that is as close as possible to what is biologically relevant, so you 
should definitely change the choices of symmetry copies to make a proper 
heterodimer in your PDB entry.  The easiest way I know to do that is with the 
option in coot: Extensions->Modelling->Symm Shift Reference Chain Here (after 
centering on an atom in a symmetry copy that you want to make the master copy).

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

> On 4 Mar 2019, at 17:27, Eze Chivi 
> mailto:ezech...@outlook.com.ar>> wrote:
>
> Dear CCP4bb community:
> My crystal is a heterodimeric complex. I solved the structure using MR with a 
> related structure (containig the dimer), using a highly automated pipeline. 
> However, the MR solution is not the dimer of biological relevance. The 
> experimentally validated dimer is formed between a protomer in the ASU and 
> one from the adjacent symmetry-related pair of molecules. Is it correct to 
> use a modelling program to assemble the "biologically correct" dimer and then 
> proceed to refinement? Or... is it need to keep the MR solution and inform in 
> the PDB header how the relevant dimer is formed? Many Thanks
>
> Ezequiel
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Mandatory mmCIF format for crystallographic depositions to the PDB

[Herman . Schreuder]

I agree, how can we punch an mmCIF file on these cards?

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edwin 
Pozharski
Gesendet: Mittwoch, 20. Februar 2019 16:19
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Mandatory mmCIF format for crystallographic 
depositions to the PDB

But my 80 symbols... :)

[https://upload.wikimedia.org/wikipedia/commons/6/65/Punch_card_Fortran_Uni_Stuttgart_%283%29.jpg]

On Wed, Feb 20, 2019 at 3:08 AM John Berrisford 
mailto:j...@ebi.ac.uk>> wrote:

From July 1st 2019 onward, mmCIF format files will become mandatory for 
crystallographic depositions to the Protein Data Bank. PDB format files will no 
longer be accepted for deposition of structures solved by these techniques.

PDBx/mmCIF will be the only format accepted for deposition of PDB structures 
resulting from macromolecular crystallography (MX), including X-ray, neutron, 
fiber, and electron diffraction methods via OneDep starting July 1st 2019. The 
deposition of PDBx/mmCIF format files will improve the efficiency of the 
deposition process and enhance validation through capture of the more extensive 
experimental metadata supported by PDBx/mmCIF, compared to the legacy PDB 
format.

For information about these changes, please visit the wwPDB news 
page.
 If you have any queries or comments regarding these changes, please contact 
the wwPDB consortium via 
deposit-h...@mail.wwpdb.org.
The wwPDB consortium



--
John Berrisford
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492529

http://www.pdbe.org
http://www.facebook.com/proteindatabank
http://twitter.com/PDBeurope




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] refmac same residue different names

[Herman . Schreuder]

Ed,

I did understand your question correctly and (at least for ligands) the 
procedure I and also Diana Tomchick described, worked. However, I just did a 
test with both Refmac and Buster and it seems that these programs have now so 
far been perfected that “errors” like this cannot occur anymore.

So it seems that the poor crystallographers who have crystallized proteins 
which are heterogeneous at certain positions, will have to switch to Phenix.

Best,
Herman


Von: Edwin Pozharski [mailto:pozharsk...@gmail.com]
Gesendet: Mittwoch, 6. Februar 2019 18:19
An: Schreuder, Herman /DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [EXTERNAL] [ccp4bb] refmac same residue different names

Herman,

thanks - however, it seems like I have poorly worded my question.  I do know 
how to generate alternate conformers, what the PDB ATOM record format is etc.  
The point was that when I have alternate conformers that carry the same residue 
ID but different residue types, Refmac exits with the error.  The question was 
whether there is a "native" solution to this that does not include some pdb 
file acrobatics (i.e. separating the alternative type into a separate residue 
and enforcing connectivity using elaborate LINK records).   Based on what I see 
so far, there likely isn't any such native option.  Whether these situations 
are common enough to warrant (possibly elaborate) software changes is a 
separate question.

Cheers,

Ed.

---
I don't know why the sacrifice didn't work. The science seemed so solid.
Julien XIII, Lord of the Lemurs

On Wed, Feb 6, 2019 at 2:36 AM 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Edwin,

I do not know whether your question has been answered already, but the answer 
is simple: you have to define alternative conformations. Easiest is to generate 
them in coot with the “add alternate conformation” option in the right panel. 
You may have to delete the original unlabeled alternative conformation first 
though.
Alternatively, if you want to keep the original coordinates, or if the 
alternative residue is different: say a Leu versus a Phe you can open the pdb 
file with an editor and generate the alternative conformation yourself:
One of the residues gets an “A” in front of the residue name, e.g. ALEU, and 
the alternative residue a “B”, say BLEU. You also have to reset the occupancies 
to 0.5 for both conformations (or different fractions which add up to one).

Good luck!
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Edwin Pozharski
Gesendet: Montag, 4. Februar 2019 22:35
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names

Belated happy 2019, everyone.

For whatever obscure reason, I need to refine a model that has two different 
residue types as alternate conformers with the same residue ID.  Presented with 
a pdb file that has such feature, Refmac fails saying this

 ERROR: in chain A residue: 443
different residues have the same number

There is an error in the input coordinate file
At least one the chains has 2 residues with the same number
Check above to see error
===> Error: Problem with coordinate file

There are several ways of getting around this I can think of.  Perhaps 
duplicate chain with strict NCS for all but the residue in question could work. 
 Perhaps adding this residue as two separate chains and then adding enough LINK 
records to keep things in place could.  Either solution here is inelegant and 
requires reformating pdb file back to sanity prior to deposition.

Is there some way to allow different geometries for alternate conformers that 
is native to Refmac?

Cheers,

Ed.

PS.  I know that phenix.refine takes the mixed name pdb file straight up.  I 
still want to be able to refine such structure with refmac (and buster, 
actually, but that's a question I already asked in the appropriate forum.


Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] refmac same residue different names

[Herman . Schreuder]

Dear Edwin,

I do not know whether your question has been answered already, but the answer 
is simple: you have to define alternative conformations. Easiest is to generate 
them in coot with the “add alternate conformation” option in the right panel. 
You may have to delete the original unlabeled alternative conformation first 
though.
Alternatively, if you want to keep the original coordinates, or if the 
alternative residue is different: say a Leu versus a Phe you can open the pdb 
file with an editor and generate the alternative conformation yourself:
One of the residues gets an “A” in front of the residue name, e.g. ALEU, and 
the alternative residue a “B”, say BLEU. You also have to reset the occupancies 
to 0.5 for both conformations (or different fractions which add up to one).

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edwin 
Pozharski
Gesendet: Montag, 4. Februar 2019 22:35
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] refmac same residue different names

Belated happy 2019, everyone.

For whatever obscure reason, I need to refine a model that has two different 
residue types as alternate conformers with the same residue ID.  Presented with 
a pdb file that has such feature, Refmac fails saying this

 ERROR: in chain A residue: 443
different residues have the same number

There is an error in the input coordinate file
At least one the chains has 2 residues with the same number
Check above to see error
===> Error: Problem with coordinate file

There are several ways of getting around this I can think of.  Perhaps 
duplicate chain with strict NCS for all but the residue in question could work. 
 Perhaps adding this residue as two separate chains and then adding enough LINK 
records to keep things in place could.  Either solution here is inelegant and 
requires reformating pdb file back to sanity prior to deposition.

Is there some way to allow different geometries for alternate conformers that 
is native to Refmac?

Cheers,

Ed.

PS.  I know that phenix.refine takes the mixed name pdb file straight up.  I 
still want to be able to refine such structure with refmac (and buster, 
actually, but that's a question I already asked in the appropriate forum.


Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /



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[ccp4bb] AW: Thoughtful remark...

[Herman . Schreuder]

For me it is easy:
If your crystal does not diffract, you have a problem with your crystal, which 
is usually very difficult to optimize
If you have no crystal in your loop, you have a problem with fishing your 
crystal, which should be much easier to optimize! 

Cheers,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von CHAVAS 
Leonard
Gesendet: Freitag, 1. Februar 2019 09:43
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Thoughtful remark...

Dear all

I had one interesting comment today at the beamline, that I would like to share 
with you for your advice on how to answer this...

Is it better to have no diffraction from a crystal you shoot, or to have no 
crystal in the loop?

Hum, difficult one...

Cheers, leo

-
Leonard Chavas
- 
Synchrotron SOLEIL
Proxima-I
L'Orme des Merisiers
Saint-Aubin - BP 48
91192 Gif-sur-Yvette Cedex
France
- 
Phone:  +33 169 359 746
Mobile: +33 644 321 614
E-mail: leonard.cha...@synchrotron-soleil.fr
-



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?

[Herman . Schreuder]

A long time ago, before siliconized coverslips became commercially available, 
we used to siliconize coverslips ourselves. It is not really that much work and 
unsiliconized cover slips should be very cheap. If you wish, I could try to 
find back the protocol.
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Rajnandani Kashyap
Gesendet: Donnerstag, 31. Januar 2019 09:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass 
coverslips for crystallization?

Dear All
I am a PhD student who requires lots of coverslips (!!) for setting up hanging 
drop crystallization. The company sells it for a huge amount. Also there is a 
wide monetary difference between a normal siliconized coverslip and a 22mm 
siliconized circle coverslips. We tried to search for an alternative companies 
but couldn't get any one who sells coverslips with the same dimensions 
(0.19-0.22mm glass thickness and 22 mm glass diameter). Is there any 
alternative company (distribution in India) from where we can buy them for a 
reasonable price?
Thanks in advance for sparing your valuable time and efforts.

Regards
Rajnandani Kashyap
India



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[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Philippe,
 
As Randy just pointed out, when twinning, pseudosymmetry and other pathologies 
come into play, things really get complicated.
I agree with what you said but for the current problem, things may be more 
complicated. 

To summarize:
- "bona fide" twinning: there are two different, intergrown crystals and the 
intensities of both crystals just add up.
- "nano scale" twinning, called statistical disorder: the two orientations are 
randomly distributed through the crystals and the diffraction of both 
conformations interferes. The conformations behave like alternate conformations.
- Twinning/pseudosymmetry/wrong unit cell etc.: Here the two conformations, 
present in the large (true) unit cell and related by crystallographic or 
noncrystallographic symmetry, may not fit in the small (false) unit cell and be 
accounted for by introducing twinning where none is present. Especially with 6 
twinning operators, the refinement programs have a lot of room to tweak around 
to reduce the R-factors.

Therefore my advice would be:
- Critically check the space group and especially how weak are the weak 
reflections discarded with the small unit cell?
- the solution you got in the small unit cell may be a subset of what is 
present in the large unit cell, so I would also try molecular replacement with 
the ensemble of molecules you got in the small unit cell.

My two cents,
Herman


-Ursprüngliche Nachricht-
Von: DUMAS Philippe (IGBMC) [mailto:p.du...@ibmc-cnrs.unistra.fr] 
Gesendet: Freitag, 11. Januar 2019 11:52
An: Schreuder, Herman /DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & 
twinning


Le Vendredi 11 Janvier 2019 09:07 CET, herman.schreu...@sanofi.com a écrit:

Dear Herman,
As far as I understood the twinning problem, what you say is only true in some 
occasions, and not in others.
If the "macroscopic" domains are so small that they are smaller than the X-rays 
coherence length, then you may do what you say because the X-rays emitted by 
the different domains can interfere.
But, if the domains become large, the X-rays emitted by the different domains 
do not interfere anymore and you have to add the weighted intensities, not the 
amplitudes, of each domain.
I hope I understood well your comment and did not "interfere negatively" with 
the thread...
Best
Philippe Dumas

> Dear Lan,
>
> Thank you for your compliment. I do not use Xtriage, so I did not bother 
> looking at the log files.
>
> What I meant to say is that with twinning, the crystal has different 
> macroscopic domains where the molecules have different orientations, say one 
> domain with orientation A and one domain with orientation B. Since these 
> domains grow on top of each other, they are usually related by a twin 
> operator similar to a crystallographic operator such as a twofold axis.
> The fourier transform of the electron density of the crystal is the 
> convolution of the fourier transform of the individual molecules with the 
> crystal lattice, with the fourier transform of the individual molecules 
> usually giving the stronger contribution. So to get a solution with a decent 
> R-factor, one must include all orientations (A, B etc.) in the model, with 
> the position of the molecules in the crystal lattice contributing less to the 
> diffraction pattern. So one can put the orientations on top of each other in 
> a small unit cell using twinning, or put them in a larger unit cell at 
> different positions using crystallographic or non-crystallographic symmetry. 
> That is what I meant be "twinning" (N)CS.
>
> Hope this makes my remark a little clearer.
>
> Best,
> Herman
>
> PS: While other BB readers may have had the same question, I have posted the 
> reply to the BB. I hope you don't mind.
>
>
> -Ursprüngliche Nachricht-
> Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu]
> Gesendet: Donnerstag, 10. Januar 2019 20:53
> An: Schreuder, Herman /DE
> Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning
>
> Dear Herman,
> I have read your insightful comments on twining and tNCS for years now, which 
> is very useful and helpful!  Thanks,
>
> For Donghyuk’s case, do you think that he really has a twinning issue?  With 
> a lower symmetry, all possible twining operators is always reported in the 
> Xtriage and did not mean a real twin existed.  His L test shows a twin 
> fraction of 0.00 in his log file.  The intensity statistics does not really 
> indicate an actually twinning.  Base on the refinement, twin law is needed to 
> get refinement going.  It looks like a twin.  I am confused...
>
> > the molecules are related by "twinning" (N)CS?
>
>
> What does this mean “ twinning (N)CS"?  Would you please kindly explain it 
> further?
>
> Thanks,
>
>
> Lan
>
>
> 
> Lan Guan, MD PhD
> Associate Professor | Department of Cell Physiology and 

[ccp4bb] AW: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Lan,

Thank you for your compliment. I do not use Xtriage, so I did not bother 
looking at the log files.

What I meant to say is that with twinning, the crystal has different 
macroscopic domains where the molecules have different orientations, say one 
domain with orientation A and one domain with orientation B. Since these 
domains grow on top of each other, they are usually related by a twin operator 
similar to a crystallographic operator such as a twofold axis. 
The fourier transform of the electron density of the crystal is the convolution 
of the fourier transform of the individual molecules with the crystal lattice, 
with the fourier transform of the individual molecules usually giving the 
stronger contribution. So to get a solution with a decent R-factor, one must 
include all orientations (A, B etc.) in the model, with the position of the 
molecules in the crystal lattice contributing less to the diffraction pattern. 
So one can put the orientations on top of each other in a small unit cell using 
twinning, or put them in a larger unit cell at different positions using 
crystallographic or non-crystallographic symmetry. That is what I meant be 
"twinning" (N)CS.

Hope this makes my remark a little clearer.

Best,
Herman

PS: While other BB readers may have had the same question, I have posted the 
reply to the BB. I hope you don't mind.


-Ursprüngliche Nachricht-
Von: Guan, Lan [mailto:lan.g...@ttuhsc.edu] 
Gesendet: Donnerstag, 10. Januar 2019 20:53
An: Schreuder, Herman /DE
Betreff: Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

Dear Herman,
I have read your insightful comments on twining and tNCS for years now, which 
is very useful and helpful!  Thanks,

For Donghyuk’s case, do you think that he really has a twinning issue?  With a 
lower symmetry, all possible twining operators is always reported in the 
Xtriage and did not mean a real twin existed.  His L test shows a twin fraction 
of 0.00 in his log file.  The intensity statistics does not really indicate an 
actually twinning.  Base on the refinement, twin law is needed to get 
refinement going.  It looks like a twin.  I am confused...

> the molecules are related by "twinning" (N)CS?


What does this mean “ twinning (N)CS"?  Would you please kindly explain it 
further?

Thanks,


Lan



Lan Guan, MD PhD
Associate Professor | Department of Cell Physiology and Molecular Biophysics
Director | Center for Membrane Protein Research

3601 4th St. MS 6551 | Lubbock, TX 79430
5A148A (Office) | (1) 806 743-3102 (Phone) | lan.g...@ttuhsc.edu (E-Mail)

http://www.ttuhsc.edu/medicine/cell-physiology-molecular-biophysics/faculty/guan/
https://www.ttuhsc.edu/centers-institutes/membrane-protein-research/


> On Jan 10, 2019, at 11:10 AM, herman.schreu...@sanofi.com wrote:
> 
> CAUTION: This email originated from outside of TTUHSC. Do not click links or 
> open attachments unless you recognize the sender and know the content is safe.
> 
> 
> Dear Donghyuk,
> 
> Unfortunately, everything is possible when NCS, twinning etc. get into the 
> game. I do not have answers, but some questions for you to think about:
> - Do you really have 6 twinning operators, or only one operator and are the 
> other operators generated by (non)crystallographic symmetry?
> - Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, 
> the last angle should be 120 and for C2, only the second angle is constrained 
> to be 90. Maybe you should check that not somewhere something went wrong with 
> the cell angles.
> - How weak are the reflections that got discarded by halving the a- and 
> b-axes? Do they have significant intensity, or is it only noise?
> - By shrinking the unit cell, you may have created artificial twinning when 
> in the large unit cell the molecules are related by "twinning" (N)CS.
> - Since you seem to have found a solution with the small unit cell, you could 
> see if you could fit this solution in the large unit cell: Process in P1 in 
> the large unit cell and use the ensemble (the complete! unit cell of your C2 
> solution, as a search model.
> - Your current solution maybe correct after all, but I would analyze it very 
> critically.
> 
> Best,
> Herman
> 
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Donghyuk Shin
> Gesendet: Donnerstag, 10. Januar 2019 11:12
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] [ccp4bb] translational NCS & twinning
> 
> Dear all,
> 
> I am having tough time with my Xtal data sets those seem to be twinned or 
> have translational NCS, and it will be greatly appreciated if you can give me 
> some advices or comments!
> 
> Data was initially processed with XDS and scaled with aimless without 
> specifying certain space group 

[ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning

[Herman . Schreuder]

Dear Donghyuk,

Unfortunately, everything is possible when NCS, twinning etc. get into the 
game. I do not have answers, but some questions for you to think about:
- Do you really have 6 twinning operators, or only one operator and are the 
other operators generated by (non)crystallographic symmetry?
- Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, the 
last angle should be 120 and for C2, only the second angle is constrained to be 
90. Maybe you should check that not somewhere something went wrong with the 
cell angles.
- How weak are the reflections that got discarded by halving the a- and b-axes? 
Do they have significant intensity, or is it only noise?
- By shrinking the unit cell, you may have created artificial twinning when in 
the large unit cell the molecules are related by "twinning" (N)CS.
- Since you seem to have found a solution with the small unit cell, you could 
see if you could fit this solution in the large unit cell: Process in P1 in the 
large unit cell and use the ensemble (the complete! unit cell of your C2 
solution, as a search model.
- Your current solution maybe correct after all, but I would analyze it very 
critically.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Donghyuk 
Shin
Gesendet: Donnerstag, 10. Januar 2019 11:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] translational NCS & twinning

Dear all,

I am having tough time with my Xtal data sets those seem to be twinned or have 
translational NCS, and it will be greatly appreciated if you can give me some 
advices or comments!

Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is 
non-origin peak after patterson analysis. (attached log) And, I could not get 
the proper MR-solution from this data sets.

Because I read that twinning and tNCS cannot be properly distinguished at high 
SG, I went down to subgroup either P32 or P6 assuming that there is twinning 
which make data set seems to have apparently high SG. (procedure was same 
XDS->aimless but I specified the SG to keep them) Then, xtriage still indicates 
there is non-origin peak as before, but found twin laws for the data sets 
(attached log).
However, I still could not get the right MR-solution.
Then, I went even further down to P3 or C2, and xtriage found more twin laws 
which is make sense because of the lower SG. (attached log) Again, I could not 
get the MR-solution.
For all the MR running above, I assumed that phaser(ccp4 module) automatically 
applied tNCS if they present. or should I have to tick on button in the expert 
parameters?

Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
During this step, I played with the threshold for indexing to follow the strong 
spot for get correct SG.
I am not sure whether this is correct or not, but by putting high threshold for 
indexing (e.g. ~15) I could index the data with C2 which has half dimension for 
a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original unit cell 
(232.533, 134.202, 182.67, 90, 90, 90).
With this, I could put 3 molecules in ASU by MR. During refinement, I felt that 
the R values were not dropping, and I applied twin refinement.
without twin refinement the R values were (0.39/0.44, work/free), and applying 
twin refinement gave me significantly better values (0.23/0.26).
Because there were 6 twin operators which may cause this huge R value drop, I 
speculate whether this is true or not.

Your comments will be greatly helpful! 

With you all the best,
Donghyuk





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[ccp4bb] AW: complex multi-crystal averaging

[Herman . Schreuder]

Dear Andy,

Best would be to convert the phaser angles (plus vector) in a matrix (plus 
vector) and use this for your transformations. The phaser people should be able 
to provide you with information how to do this, since they have to do it in 
order to produce their output files. Alternatively, you may try to find a 
transformation program which takes phaser angles as an input. I do have some 
prehistoric subroutines which do all kind of conversions between angles and 
matrices, and if nothing works, I could have a look if one of those subroutines 
could to the job.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Andrew 
Lovering
Gesendet: Mittwoch, 9. Januar 2019 17:08
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] complex multi-crystal averaging

Dear All,

I suspect the way out of this is a new crystal (!) but interested to hear any 
advice.

I have two crystal forms of a 500aa protein, vaguely tube-shaped

1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map indicates it 
is a relative of other folds (but those are not so close that they'd be a good 
guide to sequence register). I have selenomet SAD phases which helps identify 
Met positions. The 3-fold and high solvent give a great map but you wouldn't 
want to build it and buccanner and phenix think similar

2=I222, 2 copies per asu, 66% solvent. This has a cell that gives wildly 
anisotropic diffraction - ~3, 3.5, 4.3 down different axes. Not really 
rectified by staraniso. No phases

So I can cut the density out of form 1 map (using secondary structure elements 
of a rough PDB as a mask), and use phaser with this density as search model to 
find the two copies with a TFZ of about 10. The phaser map shows a bit of 
detail and the solution has placed the protomers such that they agree with a 
self-rotation function. Phenix find_ncs on phaser map similarly agrees (as I 
would expect).

Now...I have an eye on multi-crystal averaging between the two forms. BUT the 
phaser map isn't good enough to manually place the PDB used in cut-out density, 
and I can't see a straightforward way of using the angle info in phaser sol to 
perform a co-ordinate transformation (but I think ccp4bb users will put me 
right on this - I'd imagine placing the PDB into the cutout density mtz then 
using PDBset?). I tried converting the phaser mtz to a map using FFT then using 
phased TF in Molrep to place the PDB but this didn't work, complaining about 
the grid used.

One last caveat - I have multiple sets for the I222 that intriguingly differ by 
12 angstrom down a 240 ang axis: doing multi-crystal averaging between  these 
two forms achieves little when I would expect otherwise (all the NCS 
correlations are good, high initial agreement)

Like I said...fingers crossed for a new crystal form!

Andy







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[ccp4bb] AW: [ccp4bb] Experimental phasing vs molecular replacement

[Herman . Schreuder]

I think Jacob is right. As long as protein crystals contain about 10% "dark 
matter"* not accounted for in any model, we cannot fake a "true" electron 
density map and it is then not surprising that an 2mFo-DFc map is closer to a 
model-based fake map than a map based on experimental phases.
HS

*This "dark matter" causes the best Rfactors for protein crystals to be ~15% 
instead of the 5% measurement errors and may include disorder, anisotropy, 
imperfectly modelled solvent etc.



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Donnerstag, 6. Dezember 2018 04:37
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Experimental phasing vs molecular replacement

>>That said, model phases are not so bad.  In fact, in all my experiments with 
>>fake data the model-phased 2mFo-DFc map always has the best correlation to 
>>the "true" map.  If you substitute the "true" phases and use the 2mFo-DFc 
>>coefficients you actually make things worse. Counter-intuitive, but true.

I don't understand what you mean by true and fake here--can you clarify? How 
are the true map and phases generated (from an original true model, I assume?), 
and how are the fake data generated? (Also from the true model?) I am wondering 
whether there is some circular reasoning?

JPK



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Assumptions on protein purification

[Herman . Schreuder]

Dear Markus,

I believe the strong assumption in the community is that a clear single peak of 
appropriate Mw is a clear indication of pure protein, worthy intensive 
crystallization efforts. Whether it is active is another question and this 
should be measured.

For your analysis, it is not important in which buffers the protein is not 
active, but whether the protein you purified is active in the buffer (maybe 
without precipitant) you used for crystallization.

A single apo structure is usually not enough to determine the catalytic 
mechanism of an enzyme, you usually need some substrate-, transition state- 
product- (analog) structures as well. If your protein is active in the 
crystallization buffer and the ligand complexes make chemical sense, you can be 
pretty sure that you have crystallized the right conformation.

If your protein is not active in the crystallization buffer, you must 
critically analyze the structure, if it makes chemical sense and if you can 
explain the absence of activity (e.g. pH far from optimum; inhibitor bound in 
the active site). I am currently working on an enzyme who's active site loves 
all kinds of substituted and unsubstituted phosphates, sulfates etc. so it is 
not active in a wide range of buffers like phosphate, MES, MOPS, HEPES etc. 
However, the crystal structures still represent the active conformation, the 
active site is just blocked by some buffer component.

Other proteins (proteases) can only be crystallized in an inactive form, since 
active they chew themselves to pieces.

Hopes this helps,
Herman




-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Markus 
Heckmann
Gesendet: Donnerstag, 11. Oktober 2018 12:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Assumptions on protein purification

Dear all,
Not directly a ccp4 question.
I am working on a multi-domain protein with multiple catalytic
centres. Purifying in gel-filtration (Äkta) with different buffers
gives a clear distinct peak indicating pure protein. We could even
crystallise it and determine 3D structure to about 2.5 A.

Why is there a strong assumption in the community (or at least in my
limited experience) that a clear single peak of appropriate Mw is
indication of *active and folded* protein that upon
crystallisation/structure determination can describe the working of
the enzyme?

When I tested the activity of the protein using assays, I found 3 out
of 4 buffers give very poor product turnover?  Could we discount the
possibility that some 3D-structures in PDB are inactive (differently
folded) and hence may not represent active state? Are there any best
practices? Or is this dependent on the protein, especially
multi-domain proteins show such weird behaviour?

Thanks for your comments,
Markus



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Weird diffraction pattern

[Herman . Schreuder]

To me, it looks like some intergrown salt crystal.
HS

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Sam Tang
Gesendet: Dienstag, 9. Oktober 2018 13:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Weird diffraction pattern

Dear all

Hello. We recently shot a crystal (a protein with small molecule as ligand) at 
a synchrotron source and see a weird pattern. 
(https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing)

Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with 
glycerol.

At first we thought it was a protein crystal contaminated with salt but on 
second thought, the lowest resolution spot was at around 7 A, which doesn't 
make sense for a protein. So we would like to solicit your experience and 
perhaps someone may have encountered similar pattern before?

Many thanks.

Kind regards

Sam




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] R-merge is too high !!

[Herman . Schreuder]

Dear Liang,

The first thing I would do is to look through your complete scan. ADXV has an 
option to display a movie of all images. It might be that some regions of your 
scan are very weak or otherwise bad. Merging these regions with good regions 
will produce high Rmerges.
I would also check for overloads and/or ice rings. These may wreak havoc on 
your scaling which, again, may produce very high Rmerges.
I would also try some other processing program (xds, mosflm, dials….), maybe 
one of these does not suffer from the scaling problem.
You should also process maybe just the first 30 or 40 frames to see what kind 
of Rmerges come out then.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Zhang 
Foggy
Gesendet: Freitag, 28. September 2018 10:10
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] R-merge is too high !!

Dear All,

Sorry for the off-topic.

I recently collected a set of data. The diffraction spots are extremely sharp. 
However, When I used HKL3000 to scale it, I get a final resolution at 3.1A with 
overall R-merge ~0.54 (R-merge in the highest 3.2A-3.1A shell: 1.59). Then I 
solve the structure with final R value 0.19 and R free value 0.24 although I 
know this Rmerge value is totally unacceptable, and the density looks perfect.

I also tried to collect other four set of data with different crystals. 
unfortunately, all of them have same problem.

I ask one of my friend who is an expert in HKL3000, but he had no idea about 
it. Does anyone has suggestions?

Here is the scale information for your review:
Space group: P43 (I also tried P1, the Rmerge value is still similar)

Shell Lower Upper Average  Average Norm. Linear Square
 limitAngstrom   I   error   stat. Chi**2  R-fac  R-fac  Rmeas   Rpim  
CC1/2CC*
  50.00   6.6711.6 0.9 0.3  1.165  0.191  0.284  0.198  0.052  
0.975  0.994
   6.67   5.30 4.5 0.5 0.3  0.952  0.317  0.313  0.329  0.086  
0.971  0.993
   5.30   4.63 7.3 0.7 0.5  0.961  0.293  0.297  0.304  0.081  
0.975  0.994
   4.63   4.21 7.0 0.8 0.6  0.986  0.369  0.358  0.382  0.101  
0.969  0.992
   4.21   3.91 5.6 0.8 0.6  1.040  0.522  0.491  0.541  0.142  
0.955  0.988
   3.91   3.68 4.6 0.9 0.7  1.064  0.718  0.669  0.746  0.203  
0.929  0.981
   3.68   3.49 3.5 0.9 0.8  1.092  1.059  0.986  1.101  0.299  
0.882  0.968
   3.49   3.34 2.6 0.9 0.8  1.092  1.382  1.298  1.438  0.395  
0.829  0.952
   3.34   3.21 2.1 0.9 0.8  1.084  1.543  1.489  1.614  0.468  
0.772  0.933
   3.21   3.10 1.6 0.9 0.8  1.070  1.591  1.669  1.680  0.529  
0.645  0.885
  All reflections  5.0 0.8 0.6  1.048  0.538  0.487  0.559  0.153

Thank you.

Liang




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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Herman . Schreuder]

Hi Kevin, Pavel and others,

Since it seems that so far nobody answered the primary question: “Is there any 
sever available to create electron density maps for cryo-em structures?” So I 
will do it. The answer is very simple: They do not need to be created, they are 
available from the pdb!

To give an example: On the RCSB pdb web-site, I searched for entry 5vai. Then 
under the button “Download Files” I selected “Download EM Map” and downloaded 
emd_8653.map.gz. As the name suggests, this file needs to be unzipped, but this 
is trivial.

Then in Coot in the “File” pull-down menu, I select “Open Map” to load the map.
Next, you may not see anything since to contour level might be too high (when I 
load the map, the contour level is around 6 rmsd) so you have to scroll down 
the contour level to see anything. As Marin and Ian pointed out, the maps are 
very similar to regular electron density maps, probably with the exception that 
the EM “electron density” maps are influenced by the local charge density. 
However in coot they behave 100% identical and you can scroll up and down the 
contour level as you like.

Of course, if the authors did not deposit their EM-map, you cannot download it, 
but the same is true for X-ray structure factors.

Happy viewing!

Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Ian 
Tickle
Gesendet: Montag, 10. September 2018 12:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM structures.


Hi Marin

I was about to comment on that too but then I realised that Pavel is referring 
to the map _contours_  (which is what most people using a map visualisation 
program like Coot actually see).  So the contoured map does represent an 
iso-potential surface.  I'm sure Pavel is aware that the original cryo-EM maps 
are 3-dimensional objects.

Cheers

-- Ian

On Mon, 10 Sep 2018 at 10:49, Marin van Heel 
<057a89ab08a1-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Unfortunately,

The problem here lies primarily in the answer given,  not so much in the 
question asked by a newcomer:

"1) In cryo-EM maps are not electron density maps but surfaces representing 
electric potential. "

The answer appears to reflect the widespread misunderstanding that EM images 
(and hence cryo-EM maps)  only show the surfaces not the internal density of 
the complexes we study.
In my Imperial College/Leiden University  lecture notes, I have always used the 
below slide to illustrate this point.

Cheers,

Marin

[cid:part1.5DA80E33.F02E5B7D@googlemail.com]



On 10/09/2018 01:38, Pavel Afonine wrote:
Hi,

Is there any sever available to create electron density maps for cryo-em 
structures?

The questions are nonsensical. Here is why:

1) In cryo-EM maps are not electron density maps but surfaces representing 
electric potential.

2) Creating such a map is essentially carrying on from cryo-EM experiment and 
obtaining the 3D reconstruction.

Are you really sure about what you are asking for?

Or, we should create the maps from mmCIF.

mmCIF is a file format. It may contain representations of rabbits, 
boysenberries or some diffraction data. So.. how you think it may be related to 
cryo-EM, in your particular case?

I am particularly interested in those cryo-em structures with high resolution, 
like 2.6~2.8A.

Sure, all are excited about high-res cryo-EM!!!

Please give me an education.

Sure. One of available universities can do this.

Cheers,
Pavel




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--

==



Prof Dr Ir Marin van Heel



Laboratório Nacional de Nanotecnologia - LNNano

CNPEM/LNNano, Campinas, Brazil



Skype:  Marin.van.Heel

email:  
marin.vanheel(A_T)gmail.com


marin.vanheel(A_T)lnnano.cnpem.br

and:

[ccp4bb] AW: [EXTERNAL] Re: identifying bound ions

[Herman . Schreuder]

Dear Kay,

I have looked at XPAND and it looks like it is part of the O-package. Do you 
know if it can also be used stand-alone?

Best,
Herman 

-Ursprüngliche Nachricht-
Von: Kay Diederichs [mailto:kay.diederi...@uni-konstanz.de] 
Gesendet: Mittwoch, 1. August 2018 15:00
An: CCP4BB@JISCMAIL.AC.UK; Schreuder, Herman /DE
Betreff: [EXTERNAL] Re: identifying bound ions

Dear Herman,

the "water scrutinizer" option of XPAND does this -. 
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.msg.ucsf.edu_local_programs_ono_manuals_xpand-5Fman.html-23S7=DwIFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=xDNvO2p9KWi88plMQdKeBus0tAym2doLwUPdQNfYRG8=sEx3IqjhUIdjbiqizdljtA1eTlNHUhuaNKMHipOp6L4=
 

best wishes,

Kay

On Tue, 31 Jul 2018 12:39:01 +, herman.schreu...@sanofi.com wrote:

>Dear BB,
>
>I know it has been discussed some time ago, but a google search did not come 
>up with anything useful.
>
>I need a program which analyzes the bound waters and suggests whether a 
>particular water might be a chloride, calcium, sulfate, sodium or something 
>else. Preferably a program that can be run off-line (not a web server), but if 
>there is no choice, we will use a webserver as well.
>
>Thank you for your suggestions!
>Herman
>
>
>
>
>
>
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>





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[ccp4bb] identifying bound ions

[Herman . Schreuder]

Dear BB,

I know it has been discussed some time ago, but a google search did not come up 
with anything useful.

I need a program which analyzes the bound waters and suggests whether a 
particular water might be a chloride, calcium, sulfate, sodium or something 
else. Preferably a program that can be run off-line (not a web server), but if 
there is no choice, we will use a webserver as well.

Thank you for your suggestions!
Herman






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[ccp4bb] AW: COOT: adding xylose to a plant glycosylation chain

[Herman . Schreuder]

Hi Marilyn,

I have been struggling with carbohydrates in the pre Glyco module era, which 
was *not nice*. However, the following strategy works reasonably well:

1)  Get the XYP monomer with the "get monomer" command.

2)  Move the XYP manually in its electron density. This can be very 
approximate

3)  Do a real space refinement of the XYP monomer alone. This should work 
should give a decent fit of the monomer

4)  Delete one of the oxygens of the future linkage and use the "make link" 
command (extensions-modeling-make link) to link your monomer to the 
carbohydrate tree.

For buster, I had some restraints files to link carbohydrates, I do not have 
any experience with phenix refine. With some luck, just a covalent link with 
reasonably well-defined sugars would be sufficient. Changing the 
stereochemistry of a standard carbohydrate link file may also do the trick.

Good luck!
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Yoder, 
Marilyn
Gesendet: Donnerstag, 5. Juli 2018 16:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] COOT: adding xylose to a plant glycosylation chain

Hello,

I'm having problems adding XYP (beta-xylose) to my glycosylation chain in a 
plant protein.

I have a 1.87 Angstrom map, 4 molecules in the asymmetric unit.  I have two 
primary N-glycosylation sites, per protein. I am not using NCS for refinement 
or model building.

The density for the NAG-(FUC)-NAG is very clear at both sites of all molecules 
in the a.u. The BMA (beta mannose after the 2nd NAG) is clear at one of the 
glycosylation sites.  At this glycosylation site, I see good density for a MAN 
at the C3 and C6 sites of BMA.  I can see good density for a xylose at the C2 - 
it is really quite clear.

Using COOT, and the Glyco module, set to Hybrid (Plant), I have been able to 
add all the NAGs, MANs, and FUCs with a press of a mouse button.  It is a *very 
nice* function of COOT.

But.  I can't get the XYP added correctly.  I select the "add an XYP-BMA 
XYP", and it pops the XYP where I want it, but then it rapidly wiggles away.  
It is as if it is actively finding an area of no density (I am not 
exaggerating).  Selecting 'Refine Tree' does not resolve the problem.  I've 
tried manually moving the xylose close enough, but this is not working well.  
It just refines away in Phenix Refine.

For refinement, I used ReadySet to generate the restraints (apparently the 
XYP-BMA XYP does not have restraints in the standard library - is that correct?)

This is a big enough problem, I am hoping I am just doing something 'big' that 
is wrong.  I'm assuming (perhaps) it is a library problem. I would appreciate 
any suggestions.

Software details:
On Windows, WinCoot 0.8.9
On Mac, Phenix version 1.13-29998-000  (Coot is version 0.8.9)
I model build in WinCoot, refine with Phenix on the Mac

Regards,
Marilyn

___
Marilyn Yoder  |  816-235-1986   |  SCB 526 | Division of Cell Biology & 
Biophysics, University of Missouri-Kansas City




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[ccp4bb] AW: Re: [ccp4bb] disulfate bond ?

[Herman . Schreuder]

Hi Shijun,

Unfortunately, it happens very often that a ligand (in your case 
Chloramphenicol) does not bind to the protein in the crystal. If you look into 
the twilight gallery, there is a large number of crystal structures where 
people desperately tried to fit their precious ligand in what turned out to be 
solvent or buffer molecules. So it is perfectly common that you don’t see your 
Chlorampenicol.

Concerning the AcCOA, here it is very well possible that the part which is 
covalently linked via the disulfide bond, is well defined in the electron 
density, but that parts of the molecule, farther away from the link are not 
visible because they are disordered. You should also look at your protein: is 
it an enzyme? What kind of reaction does it catalyze? What kind of reaction 
products do you expect? If you are uncertain what happened, you could try to 
identify the bound molecule with mass spec.

Cheers,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Donnerstag, 5. Juli 2018 11:33
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] disulfate bond ?


Hi

Thanks for all your suggestions . It is out of our hope even though it is 
disulfide bond, so I wondering what's the biological meaning for this kind of 
disulfide bond. I used AcCOA and Chloramphenicol when I screen crystal, but I 
could only put a COA  in the election density even that the election density is 
not good enough.Any good suggestion or experience?

Best Regards

shijun
-原始邮件-
发件人:"Artem Evdokimov" 
发送时间:2018-07-04 20:01:47 (星期三)
收件人: CCP4BB@JISCMAIL.AC.UK
抄送:
主题: Re: [ccp4bb] disulfate bond ?
It seems that your CoA is one carbon out of reference. You have spotty 
difference density over the ligand. Shift it left by one carbon bond and redune 
to see if density fits better.

Artem

On Wed, Jul 4, 2018, 02:26 张士军 
<21620150150...@stu.xmu.edu.cn> wrote:

Hi all

I got a structure which has COA in it, and the SH in the tail of COA is very 
close to the SH side chain of Cys in the structure. I don't know whether it is 
disulfate bond or not? I remember they should link together if they are 
disulfate bond,am I right? so what could this be? Thanks a lot!!!

best regards

shijun





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[ccp4bb] AW: [ccp4bb] Pandda problems

[Herman . Schreuder]

Dear Nick,

Thank you for your reply. I am looking forward to test a new Pandda version 
that is hopefully compatible with the latest CCP4 version.

Concerning the error messages: I think they are very useful for developers, to 
trace back in the code what had happened, but not for users, who need 
information how to change the input files and/or parameters to fix the problem. 
However, Charles Ballard send me an email that the problem could also have been 
caused by old .pyc files and gave instructions how to remove these.

If the alignment is to get a superposition, you could consider aligning on 
sequence numbers, which is trivial and which should be identical for all data 
sets.

Best regards and have a nice weekend!
Herman




Von: Pearce, N.M. (Nick) [mailto:n.m.pea...@uu.nl]
Gesendet: Freitag, 25. Mai 2018 15:59
An: Schreuder, Herman /DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Pandda problems

Hi,

Yes, that’s not a very useful error message — sorry. I am still in the process 
of intermittently rewriting the pandda code to be …better.

I don’t think you’ve necessarily done anything wrong — pandda has not been 
extensively tested on running on series of structures that are not identical 
(we normally refine one reference structure against all datasets).

However, your timing is pretty good, because I will (today) release a new 
version of pandda that should work with the latest version of ccp4. I think it 
should fix the particular problem that you have.

I’m just running tests now, but on Monday you should be able to update your 
ccp4 to the newest update and then update your version of pandda using pip, 
following the instructions on the website 
(https://pandda.bitbucket.io).

If that doesn’t work on Monday, let me know if you have any more problems.

Thanks,
Nick


On 25 May 2018, at 14:36, 
herman.schreu...@sanofi.com wrote:

Dear bulletin board,

I am trying to run Pandda on a set of about 50 data sets. I asked our system 
manager to roll back to CCP4 update 047 and by using unique and refmac, I could 
fix the problem of a few missing low resolution reflections.

However, then Pandda crashes apparently during alignment of the structures with 
the following – for me not very helpful – error messages:

Failed to align dataset x10
Traceback (most recent call last):
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/pandda/analyse/functions.py",
 line 68, in run
alignment = model.align_to(other_hierarchy=other.hierarchy, method=method, 
require_hierarchies_identical=False)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/dataset.py",
 line 96, in align_to
self.alignment = align_structures_flexible(mov_hierarchy=self.hierarchy, 
ref_hierarchy=other_hierarchy, **kwargs)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/align.py",
 line 254, in align_structures_flexible
l_ali = align_chains_flexible(chn_mov=chn_mov, chn_ref=chn_ref, 
altlocs=altlocs, cutoff_radius=cutoff_radius)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/align.py",
 line 285, in align_chains_flexible
chn_ref_cr, chn_mov_cr = common_residues(chn_ref_cb, chn_mov_cb)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/select.py",
 line 79, in common_residues
assert (max(m_seq_1)

[ccp4bb] Pandda problems

[Herman . Schreuder]

Dear bulletin board,

I am trying to run Pandda on a set of about 50 data sets. I asked our system 
manager to roll back to CCP4 update 047 and by using unique and refmac, I could 
fix the problem of a few missing low resolution reflections.

However, then Pandda crashes apparently during alignment of the structures with 
the following - for me not very helpful - error messages:

Failed to align dataset x10
Traceback (most recent call last):
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/pandda/analyse/functions.py",
 line 68, in run
alignment = model.align_to(other_hierarchy=other.hierarchy, method=method, 
require_hierarchies_identical=False)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/dataset.py",
 line 96, in align_to
self.alignment = align_structures_flexible(mov_hierarchy=self.hierarchy, 
ref_hierarchy=other_hierarchy, **kwargs)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/align.py",
 line 254, in align_structures_flexible
l_ali = align_chains_flexible(chn_mov=chn_mov, chn_ref=chn_ref, 
altlocs=altlocs, cutoff_radius=cutoff_radius)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/align.py",
 line 285, in align_chains_flexible
chn_ref_cr, chn_mov_cr = common_residues(chn_ref_cb, chn_mov_cb)
  File 
"/DD/Applications/sb/ccp4/ccp4-7.0/lib/python2.7/site-packages/giant/structure/select.py",
 line 79, in common_residues
assert (max(m_seq_1)

[ccp4bb] AW: [ccp4bb] Native Patterson Plot

[Herman . Schreuder]

Hi Bernard,

I found a script I made a long time ago which produces a native Patterson. I 
tested it with our reasonable recent CCP4 version and it still works!

You have to copy the attached script to your linux directory and make it 
executable (type chmod +rwx makpat).
To generate a patterson e.g. from myfile.mtz, you type makpat myfile
It will generate a Patterson map , a postscript file and a log file with a 
peaklist.

Good luck!
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Bernhard 
Rupp
Gesendet: Dienstag, 8. Mai 2018 23:39
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Native Patterson Plot

Hi Fellows/Developers,

I am trying to create a native Patterson map plot, but in ccp4i on windows the 
PS plot file (*.plt) is mutilated
https://www.dropbox.com/s/f3cl2980qzws3gj/mono_15_1.plt?dl=0
(wrong orientation & partly off paper) when viewed in ghostview. I vaguely 
recall these being mapslicer files,
but starting win ccp4i mapslicer from the interface entrails the attached error 
message.

Alternatively, does one know of another way of getting a quick native Patterson 
plot?
Neither phenix nor ccp4i2 seem to offer a job menu item..

Many thanks for your help.

Best, BR
--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--



makpat
Description: makpat

[ccp4bb] AW: Re: AW: [ccp4bb] tNCS problem

[Herman . Schreuder]

Dear JiYG,

With an unknown number of 1000 aa monomers, you definitively have a challenging 
project!
In that case, you will have to do some homework before running phaser:

1)  The Matthews program should give you an indication how many monomers to 
expect.

2)  P2 is a very low symmetry space group, so it might indeed be a good 
idea to process in P1 and run MR in P1.

3)   With a lot of crashes (clashes?) you may have to truncate exposed 
loops from your model. If a number of homologous structures are available in 
the pdb, you could superimpose them, truncate the loops that are different and 
use the ensemble as a search model.

4)  How good is your search model, e.g. what is the %identity or homology? 
If the model is quite remote, MR might be difficult.

In the CCP4, there is now a whole collection of automatic MR packages: Molrep, 
MrBUMP, Balbes, AMPLE, MoRDA, SIMBAD etc. you could try these as well. It will 
mainly take CPU time, not your time.

Good luck!
Herman

Von: 苏纪勇 [mailto:sujy...@nenu.edu.cn]
Gesendet: Dienstag, 8. Mai 2018 17:03
An: Schreuder, Herman /DE
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: AW: [ccp4bb] tNCS problem

Dear Herman, I tried phaser. It worked. But there are a lot of crashes in the 
structure. Meanwhile, R factors could not be lowered. I tried to use P1 to 
process the data, but I do not know how many monomers in the asymetric unit. 
Bests, JiYG
On 05/08/2018 22:50, 
herman.schreu...@sanofi.com wrote:
Dear JiYG,

Unless the tNCS has caused processing problems, Phaser should automatically 
deal with tNCS and I would recommend to just give it a try. If it fails, you 
could try more sophisticated approaches.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Dienstag, 8. Mai 2018 16:41
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] tNCS problem

Dear all, Recently, I collected a data set of a crystal of a big protein, which 
contains 1000 amino acids. I processed the data to P2. But the data set has 
tNCS problem. I want to do molecular replacement. Is there anyone know how to 
deal with this problem? Thanks, JiYG

[ccp4bb] AW: [ccp4bb] tNCS problem

[Herman . Schreuder]

Dear JiYG,

Unless the tNCS has caused processing problems, Phaser should automatically 
deal with tNCS and I would recommend to just give it a try. If it fails, you 
could try more sophisticated approaches.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Dienstag, 8. Mai 2018 16:41
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] tNCS problem

Dear all, Recently, I collected a data set of a crystal of a big protein, which 
contains 1000 amino acids. I processed the data to P2. But the data set has 
tNCS problem. I want to do molecular replacement. Is there anyone know how to 
deal with this problem? Thanks, JiYG

[ccp4bb] AW: [ccp4bb] determining the point group and the space group

[Herman . Schreuder]

Hi Gihan,

I guess your XFEL data set consists of data of hundreds of micro-crystals with 
only partials?

The first thing I would do is to ask the beamline people for the best strategy. 
Since they (should) have tested the beamline, they are probably the best people 
to ask.

Next, in the early stage I would not worry about the point group and space 
group but try to process all images in P1. Depending of the crystal 
orientation, only two cell axis might be well defined in the individual images. 
From all these individual images, it should be possible to distill the cell 
lengths and angles. Using this parameters, I would again process all images and 
compile a P1 data set.

Then, I would analyze the resulting data set with pointless or aimless or 
similar programs to try to find out what the point group and space group COULD 
be.

Good luck!
Herman  

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Gihan 
Ketawala
Gesendet: Donnerstag, 19. April 2018 03:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] determining the point group and the space group

Hi,
my question is;
how should one determine a point group and the space group of an unknown 
crystal?

I have a protein crystal with know unit-cell parameters. (these are XFEL data 
so indexing wouldn't give the point and space groups). I checked the PDB, but 
no luck the PDB structures have the different space group assigned, no 
definitive answer hopefully, somebody can point me in the right direction 

Best,
Gihan

[ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[Herman . Schreuder]

PS: If you have two different home-brewn ligands, you have to rename one of 
them (pdb and cif), otherwise the same dictionary will be applied to two 
different ligands. Also make sure your cif file is a dictionary and not just a 
coordinate file.
HS

Von: Schreuder, Herman /DE
Gesendet: Montag, 5. März 2018 14:31
An: 'Colin Levy'; CCP4BB@JISCMAIL.AC.UK
Betreff: AW: [ccp4bb] Small molecule not refined in coot.

Hi Colin and Michel,

In my experience, both refmac and coot will use the most recently read-in cif 
dictionary and there is no need to try to find an unique identifier for each 
new ligand one uses. The new dictionary overrides the old one. Finding a unique 
identifier for each new ligand would be a terrible and unnecessary hassle.

What usually goes wrong is that the name of the residue in the pdb file does 
not match the name of the residue in the cif file. And the same holds for the 
atom names. I would check with an editor that the resdue and atom names of the 
pdb file do match the residue and atom names in the cif dictionary.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Colin 
Levy
Gesendet: Montag, 5. März 2018 13:38
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Small molecule not refined in coot.

Hi Michel,

If your ligand is designated as DRG in your pdb then refinement programs will 
anticipate that it is:

Chemical Description
Name

5,6-DIHYDRO-BENZO[H]CINNOLIN-3-YLAMINE

Formula

C12 H11 N3

Formal charge

0

Molecular weight

197.236 g/mol

Component type

NON-POLYMER



If this is not the case then you need to choose a unique identifier, this 
should then allow your cif library to be utilised appropriately during 
refinement.

Colin


[cid:image001.png@01D3B48F.5E6EF790]

Dr. Colin W. Levy
MIB G016
Tel.  0161 275 5090
Mob.07786 197 554
c.l...@manchester.ac.uk

 Manchester Institute of Biotechnology | University of Manchester | 3.020 
Garside Building | 131 Princess Street | Manchester | M1 7DN

[cid:image002.png@01D3B48F.5E6EF790][cid:image003.png@01D3B48F.5E6EF790][cid:image004.png@01D3B48F.5E6EF790][cid:image005.png@01D3B48F.5E6EF790][cid:image006.png@01D3B48F.5E6EF790]










On 5 Mar 2018, at 12:30, M T > 
wrote:

Dear all,
I am actually dealing with a structure containing an unnatural ligand.
I generated the pdb file by drawing it on PRODRG server, I did a manual pre-fit 
in the map using coot and I manually merged the pdf file of my protein with the 
one of the ligand.
After that I did few cycles of refinement using Refmac5, with my mtz, my merged 
pdb file and the cif library of the ligand as input files.
First I saw that even if Refmac "completed succesfully" it didn't modify 
coordinates of the ligand, second I saw a warning about the "DRG" molecule in 
the log of Refmac (WARNING: duplicated name of monomer DRG Last entry will be 
used.), third in Coot I cannot use "Real Space Refine Zone" with the ligand and 
when I try to import the CIF dictionary produced by PRODRG (through the server 
or even the 

[ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[Herman . Schreuder]

Hi Colin and Michel,

In my experience, both refmac and coot will use the most recently read-in cif 
dictionary and there is no need to try to find an unique identifier for each 
new ligand one uses. The new dictionary overrides the old one. Finding a unique 
identifier for each new ligand would be a terrible and unnecessary hassle.

What usually goes wrong is that the name of the residue in the pdb file does 
not match the name of the residue in the cif file. And the same holds for the 
atom names. I would check with an editor that the resdue and atom names of the 
pdb file do match the residue and atom names in the cif dictionary.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Colin 
Levy
Gesendet: Montag, 5. März 2018 13:38
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Small molecule not refined in coot.

Hi Michel,

If your ligand is designated as DRG in your pdb then refinement programs will 
anticipate that it is:

Chemical Description
Name

5,6-DIHYDRO-BENZO[H]CINNOLIN-3-YLAMINE

Formula

C12 H11 N3

Formal charge

0

Molecular weight

197.236 g/mol

Component type

NON-POLYMER



If this is not the case then you need to choose a unique identifier, this 
should then allow your cif library to be utilised appropriately during 
refinement.

Colin


[cid:image006.png@01D3B48E.20BC8DA0]


Dr. Colin W. Levy
MIB G016
Tel.  0161 275 5090
Mob.07786 197 554
c.l...@manchester.ac.uk

 Manchester Institute of Biotechnology | University of Manchester | 3.020 
Garside Building | 131 Princess Street | Manchester | M1 7DN

[cid:image007.png@01D3B48E.20BC8DA0][cid:image008.png@01D3B48E.20BC8DA0][cid:image009.png@01D3B48E.20BC8DA0][cid:image010.png@01D3B48E.20BC8DA0][cid:image011.png@01D3B48E.20BC8DA0]












On 5 Mar 2018, at 12:30, M T > 
wrote:

Dear all,
I am actually dealing with a structure containing an unnatural ligand.
I generated the pdb file by drawing it on PRODRG server, I did a manual pre-fit 
in the map using coot and I manually merged the pdf file of my protein with the 
one of the ligand.
After that I did few cycles of refinement using Refmac5, with my mtz, my merged 
pdb file and the cif library of the ligand as input files.
First I saw that even if Refmac "completed succesfully" it didn't modify 
coordinates of the ligand, second I saw a warning about the "DRG" molecule in 
the log of Refmac (WARNING: duplicated name of monomer DRG Last entry will be 
used.), third in Coot I cannot use "Real Space Refine Zone" with the ligand and 
when I try to import the CIF dictionary produced by PRODRG (through the server 
or even the one generated through CCP4 ProDrg), it causes Coot crash (** 
(coot-bin:4467): WARNING **: Widget not found: 

[ccp4bb] AW: [ccp4bb] validating a homlology model

[Herman . Schreuder]

Dear Careina,

If you do not have coordinates, what do you have then? A sequence alignment? In 
that case you can look at the %identity and %homology to see how well the known 
structure fits the unknown structure.

If you do have coordinates in some other format generated by some modeling 
software, you have to convert these coordinates into pdb format. Most modeling 
program do have a “save as” or “export” option to generate a file in pdb 
format. If that fails, there are also all kind of format converting programs, 
but then you need to tell us the format you have right now.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina 
Edgooms
Gesendet: Freitag, 2. März 2018 12:44
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] validating a homlology model

Dear all

What programs are best used for validate homology models? I know of molprobity 
but if there are no coordinates I cannot use it. Is there a way to use such 
programs with homology models?

Also I wish to use pdbepisa for to charaterise dimer interface but again for 
homology model this cannot be done as there is no PDB model. Does anybody know 
way to use PISA software on my own model that is not deposited in PDB?

Thank you in advance
Careina

[ccp4bb] AW: [ccp4bb] Publishing structure of a protein

[Herman . Schreuder]

Dear Raj,

The pdb will accept any crystal structure submitted to them. They do not have 
editors and referees that refuse to accept crystal structures. In the worst 
case, your structure may have validation problems and you will get questions 
about it. But this will happen with published crystal structures as well.

There are online journals that will accept any paper (even fake ones) as long 
as the submission fee is paid. However, for “real” journals, it will depend on 
the information you get from your complex: does the buffer component bind in 
the active site? Could it be used as a kind of substrate/natural ligand analog 
to derive conclusions about the biological function of your protein? In that 
case you should include a minimum of biochemical data, showing that the buffer 
compound is a (competitive) inhibitor/activator of the protein, even if it is a 
very weak one.

However, if your buffer component binds at some random place on the protein 
surface, or at some crystal contact, it may well be an artifact and it might be 
difficult to get such a structure published. Concerning the journal, look for 
journals that publish similar papers on similar proteins. There you have the 
best chance to get your paper accepted.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von raj kumar
Gesendet: Montag, 29. Januar 2018 07:39
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Publishing structure of a protein

Hi,
We purified a protein whose structure (apo) is already known in pdb database 
and determined the crystal structure with a component of buffer. Can we submit 
this structure in pdb database and publish it as a research paper (only 
structure)?  Also can anyone suggest some journals for this?
Thanks
Raj

[ccp4bb] AW: Re: [ccp4bb] coordinate transformation

[Herman . Schreuder]

If you use coot with on the fly map calculation (e.g. you load an mtz and not a 
map file), you do not need to transform the map. Otherwise I would recommend to 
run one more round of refinement and produce a new map your usual way. This 
will also get rid of any rounding errors due to the transformation.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu
Gesendet: Montag, 18. Dezember 2017 14:16
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] coordinate transformation

Dear All,

If I have a set of PDB with the corresponding density map, after I transform 
the PDB based on the suggestion of everybody, is any way to transform the map 
so that the map will be fit with the transformed PDB?

Smith




At 2017-12-18 18:39:34, "Eleanor Dodson" 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:

I showed you pdbset ..
Find the centre of mass for your assembly.
Move it where you will
pdbset xyzin mow.pdb
end
Find  CoM 0.7 1.3 -0.2
Hmm - a little thought - centre at 1 -1 0   say
pdbset yzin now.pdb xyzout changed.pdb
symgen x , y-2, z
end
New CoM  0.7 -0.7  -0.2
Eleanor



On 18 December 2017 at 00:19, Edward A. Berry 
> wrote:
Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? or 
0.1,0.1,0.1 if I still want it near the origin but biased toward the inside of 
the positive-going cell?
eab


On 12/14/2017 07:23 PM, James Holton wrote:
What I usually do for this is make a copy of the PDB file and change all the 
atom x-y-z positions to "1.000".  Then I use something like reforigin or my 
"origins.com"
 script to shift the original coordinates via allowed symmetry operations, 
origin shifts, or perhaps indexing ambiguities until it is as close as possible 
to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 0,0,0 because 
there are generally at least two symmetry-equivalent places that are 
equidistant from the origin. Declaring the reference to be a bit off-center 
breaks that ambiguity, and also biases the result toward having all-positive 
x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:

Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...


Thanks,

Tommi

[ccp4bb] AW: Re: [ccp4bb] Van der waals force

[Herman . Schreuder]

Hi Jiri,

A low-tech solution that will certainly work, is just to manually show the 
relevant distances. In coot under measure there is an option to show distances, 
just by clicking on the two atoms involved.

Good luck!
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von chemocev 
marker
Gesendet: Samstag, 9. Dezember 2017 16:57
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Van der waals force

Hi
Thanks for your good help and I am wondering is there clear way to show these 
forces like H-bond. I tried KING and can see the VDW radii but not the visual 
representation. Does coot has any option for this.
best
Jiri

On Fri, Dec 8, 2017 at 8:57 AM, HERMAN VAN TILBEURGH 
> wrote:
jiri, VDW forces are always acting and between any pair of atoms
the optimal distance (most favourable interaction energy) depends on the pair 
of atoms involved in the interaction, but is a big bitter than the sum of 
atomic radii
all the best
herman
Herman van Tilbeurgh
Professor structural biology
Directeur Adjoint Ecole Doctorale Innovation Thérapeutique: du fondamental à 
l'appliqué

Institut de Biologie Intégrative de la Cellule - I2BC
UMR 9198 CNRS- Université Paris Sud
Team: Fonction et Architecture des Assemblages MacroMoléculaires
http://www.i2bc.paris-saclay.fr/spip.php?article256

Batiment 430
91405 Orsay
France

Tel: 33 1 69 15 31 55
fax: 33 1 69 85 37 15
herman.van-tilbeu...@u-psud.fr





Le 8 déc. 2017 à 08:52, KLAHOLZ bruno 
> a écrit :


Hello,

van der Waals interactions are very weak, this is why we usually speak about 
van der Waals contacts rather than interactions.
These are usually in the range of 3.5-3.8/4 Å (smaller than that may indicate a 
close contact or steric clash of an atomic model under refinement), 
corresponding to the packing of the van der Waals spheres of the individual 
atoms.
In hydrogen bond interactions, the term “interaction” normally implies sharing 
a hydrogen atom between two polar residues, for example between the hydroxyl 
group of a threonine side chain with a carbonyl group of the main chain peptide 
backbone; in there one should take into account the geometry as well (e.g. 
~120°-180° is favorable, 90° is not). Note that some positions such as Calpha-H 
can be slightly polarized (these contribute to bifurcated H-bonds in beta 
sheets for example, see e.g. 
https://www.ncbi.nlm.nih.gov/pubmed/12220491
 ).
In the context of series of van der Waals contacts between hydrophobic residues 
there can be additive effects of the weak interactions with then sum up, but in 
this context one should also consider entropic effects such as de-solvatation 
which becomes favorable energetically.

Hope this helps.

Best regards,

Bruno


###
Bruno P. Klaholz
Centre for Integrative Biology
Institute of Genetics and of Molecular and Cellular Biology
67404 ILLKIRCH
FRANCE
http://igbmc.fr/Klaholz




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of chemocev 
marker
Sent: 08 December 2017 07:55
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Van der waals force

Hi
I just have a basic question if the Van der waals interaction will exist 
between the hydrophobic residues or it can also be contributed by the polar 
residues as well. What distance is required for the Van der waals interaction.
best
Jiri

[ccp4bb] AW: Differences in a homodimer protein

[Herman . Schreuder]

Dear Denis,

I would first superimpose both monomers to see if you can find a reason why one 
subunit has a bound water and the other not, which would in general be flanking 
side chains in (slightly) different positions. Next I would look for some 
global differences between the subunits that could be linked to crystal 
contacts explaining the non-random distribution of the subunits. However, these 
differences might be quite subtle and difficult to detect.

As Emily suggested, you could also do some functional assay's to see if there 
is any positive (or negative) cooperativity between the subunits providing 
independent support for functional differences between the subunits.

My 2 cts,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Denis 
Rousseau
Gesendet: Dienstag, 28. November 2017 20:04
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein

Hi BB members

I have a homodimeric protein, which contains metal centers. In several 
different derivatives I find a water molecule on a metal center in one subunit 
which is absent on the other.  It is always the same subunit that contains the 
water molecule. The resulution is ~2.4 A. Is this an artifact or a functional 
difference? If it is truly homodimeric I would expect differences to be random. 
The space group is P212121.

Thanks for the advice.

Denis Rousseau

[ccp4bb] AW: [ccp4bb] Spacegroup for a symmetric monomeric protein

[Herman . Schreuder]

Dear Noguchi,

If I understand correctly, you have one protein with 8 identical repeats and a 
few additional residues at the N-terminus that you cannot see in the electron 
density map. This means, that the N-terminal repeat is different from the other 
repeats and theoretically, you should submit the complete molecule in the 
lower-symmetry space group, including data processed at lower symmetry.

However, since the N-terminal residues are not visible, I suspect that they are 
not involved in any crystal contacts and that your protein molecules have 
random orientations within the 8-fold internal symmetry, averaging-out any 
differences between the different repeats. In this case, I would submit the 
partial molecule in the high-symmetry space group, but it will probably be 
something the pdb has not foreseen and you may have to do some explanation.

Concerning the lower Rfree in your lower symmetry space group: With 
non-crystallographic symmetry, you have to carefully select your free 
reflections (including all NCS mates), otherwise your free reflections will not 
be free, but coupled via NCS to your working set reflections, which I suspect 
will be the case for you. 

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Hiroki 
Noguchi
Gesendet: Montag, 27. November 2017 13:30
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Spacegroup for a symmetric monomeric protein

Dear All,

We have a rather confusion situation currently with our designer proteins. They 
are highly symmetric and consist of identical amino acid repeats in tandem that 
are organized in 3D around a central (rotation axis). Our first series of 
proteins like this was the Pizza
(3ww9) which crystallized in P212121.

However for other proteins we have now a bit of a problem. After 
crystallization of our new proteins some of them crystallized in such a way 
that we can solve the structure with such a symmetry that only a fragment of 
the protein is present. For instance we only see 2 repeats in the asymmetric 
unit but in fact in the monomeric structure there should be 8. The 8fold 
symmetric protein can be reconstructed from crystal symmetry.

My question is now. Should we solve and deposit this crystal structure to the 
most symmetrical smallest asymmetric unit resulting in an incomplete protein in 
the asymmetric unit or should we solve the structure in a lower space group 
such that the asymmetric unit contains a full length monomeric protein. The 
protein has additional few amino acids at N-terminal but we can not see these 
regions on electronic density map. When comparing the two R free we see that 
the lowest spacegroup has a slightly better (4%) R free value than the higher 
spacegroup with an incomplete protein in the asymmetric unit.

The PDB document
(https://urldefense.proofpoint.com/v2/url?u=https-3A__cdn.rcsb.org_wwpdb_docs_documentation_annotation_wwPDB-2DA-2D2017Aug02-2DV2.8.pdf=DwIBaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=1Y5lSTX28-d9UR3haS7N8B16DvGd8reLwqeZwMRY3yg=mUPJXE8ewFueTn3Jfbg-QgxnjguFB5i3CjJvQz1v08Q=
 ) is described, "The asymmetric unit may be one molecule or one subunit of a 
multimeric protein, but it can also be more than one." on page 34.

So should we go for the highest symmetry as observed from the diffraction data, 
or go for a lower symmetry agreeing with a full protein.



Thank you for you help.



Kindest Regards,
Hiroki noguchi
KU LEUVEN, Belgium

[ccp4bb] AW: [ccp4bb] Scripting for COOT

[Herman . Schreuder]

Dear Martin,

I use a script which invokes the coot command similar to what you describe with 
--script mapcent added on the same line. The mapcent script is attached. You 
can either go to a certain atom, or set hte rotation center in Å coordinates. 
You can add whatever commands to the script to get the view and options you 
like.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Martín 
Martínez Ripoll
Gesendet: Donnerstag, 16. November 2017 12:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Scripting for COOT

Dear all,

I am trying to write a long script that, among others, runs COOT, and for this 
purpose we use something like:

coot  --pdb  refmac-out.pdb--auto refmac-out.mtz

However, I do not know how to include in the script an instruction or keyword 
to centre at a particular residue number.
Does anybody know how to do it?

Thanks in advance,
Martin
_
Dr. Martin Martinez-Ripoll
Research Professor Emeritus
xmar...@iqfr.csic.es
Department of Crystallography & Structural Biology
www.xtal.iqfr.csic.es
www.xtal.iqfr.csic.es/Cristalografia/
Telf.: +34 917459550
Consejo Superior de Investigaciones Científicas
Spanish National Research Council
[cid:image001.jpg@01D35EFD.59627D40]



mapcent
Description: mapcent

[ccp4bb] AW: [ccp4bb] High R/Rfree

[Herman . Schreuder]

Dear Radhika,

What reason does Xtriage give for declaring the reflections to be outliers? Too 
weak, too strong, other reasons? As was mentioned before, what is the 
resolution of your data? In cases like this, it is always good to have a look 
at the diffraction images to see if there is some problem there like streaks, 
ice rings etc.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Radhika 
Singh
Gesendet: Dienstag, 14. November 2017 00:45
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] High R/Rfree

Hello All,

I am currently working on the structure of a DNA protein complex.  The data has 
been processed in space group P1 (53.042   59.527   78.526 105.24  98.03 106.99 
P 1, Rpim 11.7%).  At this stage I have almost 85% model is complete but my 
R/Rfree is stuck as 26%/34%.

I have some concerns and questions:
* Xtriage says there are a large number of outliers; however no 
pseudotranslational symmetry is detected by the program.  What are the other 
reasons for outliers?

* I am trying phenix.refine for refinement with the default settings. Is there 
any special setting that can help me?

I would like to have some suggestions about my problem.

Thanks in advance

Radhika

[ccp4bb] AW: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Herman . Schreuder]

Dear Jacob,

The big advantage of microscopes (whether using electrons or X-rays) is of 
course that you 1) do not need crystals and 2) get phase information. However, 
aligning an extremely large number of single molecule images is non-trivial and 
this is the reason it is still very hard, if not impossible, to use this 
technique for molecules with a mw of less than 100 kDa. 

Also, the X-ray image of a single molecule would be extremely weak and I am not 
sure current technology would be able to record such an image.

Crystals have billions of molecules, almost perfectly aligned which produce 
very good electron density maps. It is just that many proteins are very 
difficult if not impossible to crystallize that makes cryoEM so popular.

Best,
Herman 


 

-Ursprüngliche Nachricht-
Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org] 
Gesendet: Freitag, 10. November 2017 16:55
An: Schreuder, Herman /DE; CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] RE: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging 
Conundrum

"Quality of image" has a lot of parameters, including resolution, noise, 
systematic errors, etc. I am not aware of a global "quality of image" metric.

One other consideration, not related to your comment: imagine if we had an 
x-ray lens through which we could take confocal images of a protein molecule or 
crystal, output as a voxel array. Would we really still prefer to measure 
diffraction patterns rather than the equivalent real space image, even assuming 
we had some perfect way to solve the phase problem? Or conversely, should we 
try to do fluorescence imaging in diffraction mode, due to its purported 
information efficiency?

JPK

-Original Message-
From: herman.schreu...@sanofi.com [mailto:herman.schreu...@sanofi.com]
Sent: Friday, November 10, 2017 10:22 AM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

At the bottom line, it is the quality of the image, not only the amount of 
pixels that counts. Adding more megapixels to a digital camera with a poor lens 
(as some manufacturers did), did not result in any sharper or better images.
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Freitag, 10. November 2017 15:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging 
Conundrum

It seems, then, to be generally agreed that the conversion between voxels and 
Fourier terms was valid, each containing the same amount of information, but 
the problem was in the representation, and there was just trickery of the eye. 
I was thinking and hoping this would be so, since it allows a pretty direct 
comparison of crystal data to microscopic imaging data. I guess a litmus test 
would be to decide whether a voxel version of the electron density map would 
work equivalently well in crystallographic software, which I suspect it would. 
If so, then the same techniques--so effective in extracting information for the 
relatively information-poor crystal structures--could be used on fluorescence 
imaging data, which come in voxels.

Regarding information-wealth, in Dale's example, the whole hkl set was 4.1 MB. 
One frame in a garden-variety XYZT fluorescence image, however, contains about 
2000 x 2000 x 100 voxels at 16-bit, i.e., 400 million bits or 50 MB. In some 
data sets, these frames come at 10 Hz or more. I suspect that the I/sigma is 
also much better in the latter. So, with these data, and keeping a 
data:parameters ratio of ~4, one could model about 100 million parameters. This 
type of modelling, or any type of modelling for that matter, remains almost 
completely absent in the imaging world, perhaps because the data size is 
currently so unwieldy, perhaps also because sometimes people get nervous about 
model biases, perhaps also because people are still improving the imaging 
techniques. But just imagine what could be done with some crystallography-style 
modelling!

Jacob Keller



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan 
Croll
Sent: Friday, November 10, 2017 8:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

Or a nice familiar 2D example: the Ramachandran plot with 7.5 degree binning, 
as a grid (left) or with bicubic smoothing (right). Different visualisations of 
the same data, but the right-hand image uses it better.

On 2017-11-10 08:24, herman.schreu...@sanofi.com wrote:
> In line with Dale's suggestions, I would suggest that you reformat 
> your voxel map into the format of an electron density map and look at 
> it with coot. I am sure it will look much better and much more like 
> the electron density we are used to look at. Alternatively, you could 
> display an bona fide electron density map as 

[ccp4bb] AW: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Herman . Schreuder]

At the bottom line, it is the quality of the image, not only the amount of 
pixels that counts. Adding more megapixels to a digital camera with a poor lens 
(as some manufacturers did), did not result in any sharper or better images.
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Freitag, 10. November 2017 15:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging 
Conundrum

It seems, then, to be generally agreed that the conversion between voxels and 
Fourier terms was valid, each containing the same amount of information, but 
the problem was in the representation, and there was just trickery of the eye. 
I was thinking and hoping this would be so, since it allows a pretty direct 
comparison of crystal data to microscopic imaging data. I guess a litmus test 
would be to decide whether a voxel version of the electron density map would 
work equivalently well in crystallographic software, which I suspect it would. 
If so, then the same techniques--so effective in extracting information for the 
relatively information-poor crystal structures--could be used on fluorescence 
imaging data, which come in voxels.

Regarding information-wealth, in Dale's example, the whole hkl set was 4.1 MB. 
One frame in a garden-variety XYZT fluorescence image, however, contains about 
2000 x 2000 x 100 voxels at 16-bit, i.e., 400 million bits or 50 MB. In some 
data sets, these frames come at 10 Hz or more. I suspect that the I/sigma is 
also much better in the latter. So, with these data, and keeping a 
data:parameters ratio of ~4, one could model about 100 million parameters. This 
type of modelling, or any type of modelling for that matter, remains almost 
completely absent in the imaging world, perhaps because the data size is 
currently so unwieldy, perhaps also because sometimes people get nervous about 
model biases, perhaps also because people are still improving the imaging 
techniques. But just imagine what could be done with some crystallography-style 
modelling!

Jacob Keller



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan 
Croll
Sent: Friday, November 10, 2017 8:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

Or a nice familiar 2D example: the Ramachandran plot with 7.5 degree binning, 
as a grid (left) or with bicubic smoothing (right). Different visualisations of 
the same data, but the right-hand image uses it better.

On 2017-11-10 08:24, herman.schreu...@sanofi.com wrote:
> In line with Dale's suggestions, I would suggest that you reformat 
> your voxel map into the format of an electron density map and look at 
> it with coot. I am sure it will look much better and much more like 
> the electron density we are used to look at. Alternatively, you could 
> display an bona fide electron density map as voxel blocks and I am 
> sure it will look similar to the voxel map you showed in your first 
> email.
> 
> Best,
> Herman
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Dale Tronrud
> Gesendet: Freitag, 10. November 2017 08:08
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] Re: [ccp4bb] Basic Crystallography/Imaging 
> Conundrum
> 
>Ethan and I apparently agree that anomalous scattering is "normal"
> and Friedel's Law is just an approximation.  I'll presume that your 
> "unique" is assuming otherwise and your 62,500 reflections only 
> include half of reciprocal space.  The full sphere of data would 
> include 125,000 reflections.  Since the cube root of 125,000 is 50 you 
> get a range of indices from -25 to +25 which would give you 2 A 
> resolution, which is still far from your hope of 1 A.
> 
>For your test case of 1 A resolution with 50 A cell lengths you 
> want your indices to run from -50 to +50 giving a box of reflections 
> in reciprocal space 101 spots wide in each direction and a total of
> 101^3 =
> 1,030,301 reflections. (or 515,150.5 reflections for your Friedel 
> unique with the "half" reflection being the F000 which would then be 
> purely real valued.)
> 
>Assuming you can fit your structure factors into 16 bits (You had 
> better not have many more than 10,000 atoms if you don't want your
> F000 to overflow.) the information content will be 1,030,301 * 2 * 16 
> bits (The "2" because they are complex.) giving 32,969,632 bits.
> 
>If you spread this same amount of information across real space you 
> will have 1,030,301 complex density values in a 50x50x50 A space 
> giving a sampling rate along each axis of 101 samples/unit cell.
> 
>Complex density values?  The real part of the density is what we 
> call the electron density and the imaginary part we call the anomalous 
> density.  If there is no anomalous scattering then Friedel's Law 

[ccp4bb] AW: Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

[Herman . Schreuder]

In line with Dale's suggestions, I would suggest that you reformat your voxel 
map into the format of an electron density map and look at it with coot. I am 
sure it will look much better and much more like the electron density we are 
used to look at. Alternatively, you could display an bona fide electron density 
map as voxel blocks and I am sure it will look similar to the voxel map you 
showed in your first email.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dale 
Tronrud
Gesendet: Freitag, 10. November 2017 08:08
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Basic Crystallography/Imaging Conundrum

   Ethan and I apparently agree that anomalous scattering is "normal"
and Friedel's Law is just an approximation.  I'll presume that your "unique" is 
assuming otherwise and your 62,500 reflections only include half of reciprocal 
space.  The full sphere of data would include 125,000 reflections.  Since the 
cube root of 125,000 is 50 you get a range of indices from -25 to +25 which 
would give you 2 A resolution, which is still far from your hope of 1 A.

   For your test case of 1 A resolution with 50 A cell lengths you want your 
indices to run from -50 to +50 giving a box of reflections in reciprocal space 
101 spots wide in each direction and a total of 101^3 =
1,030,301 reflections. (or 515,150.5 reflections for your Friedel unique with 
the "half" reflection being the F000 which would then be purely real valued.)

   Assuming you can fit your structure factors into 16 bits (You had better not 
have many more than 10,000 atoms if you don't want your F000 to overflow.) the 
information content will be 1,030,301 * 2 * 16 bits (The "2" because they are 
complex.) giving 32,969,632 bits.

   If you spread this same amount of information across real space you will 
have 1,030,301 complex density values in a 50x50x50 A space giving a sampling 
rate along each axis of 101 samples/unit cell.

   Complex density values?  The real part of the density is what we call the 
electron density and the imaginary part we call the anomalous density.  If 
there is no anomalous scattering then Friedel's Law holds and the number of 
unique reflections is cut in half and the density values are purely real valued 
- The information content in both spaces is cut in half and they remain equal.

   By sampling your unit cell with 101 samples their rate is half that of the 
wavelength of the highest frequency reflection.  (e.q. a sampling rate of 0.5 A 
for 1 A resolution data)  This is, of course, the Nyquist Theorem which states 
that you have to sample at twice the frequency of the highest resolution 
Fourier coefficient.

  This is exactly how an FFT works.  It allocates the memory required to store 
the structure factors and it returns the map in that same array - The number of 
bytes is unchanged.  It also guarantees that the calculation is reversible as 
no information is lost in either direction.

   So, why does your blocky image look so bad?  First you have sampled too 
coarsely.  You should have twice the sampling rate in each direction.

   The next point is more subtle.  You are displaying each voxel as a block.  
This is not correct.  The sharp lines that occur at the boundaries between the 
blocks is a high frequency feature which is not consistent with a 1 A 
resolution image.  Your sample points should be displayed at discrete points 
since they are not the average density within a block but the value of the 
density at one specific point.

   What is the density of the map between the sampled points?  The Fourier 
series provides all the information needed to calculate them and you can 
calculate values for as fine a sampling rate as you like, just remember that 
you are not adding any more information because these new points are correlated 
with each other.

   If you have only the samples of a map and want to calculate Fourier 
coefficients there are many sets of Fourier coefficients that will reproduce 
the sampled points equally well.  We specify a unique solution in the FFT by 
defining that all reflections of resolution higher than 1 A must be identically 
equal to zero.  When you calculate a map from a set of coefficients that only 
go to 1 A resolution this is guaranteed.

   When you are calculating coefficients from any old map you had better ensure 
that the map you are sampling does not contain information of a higher 
resolution than twice your sampling rate.  This is a problem when calculating 
Fcalc from an atomic model.  You calculate a map from the model and FFT it, but 
you can't sample that map at 1/2 the resolution of your interest.  You must 
sample that map much more finely because an atomic model implies Fourier 
coefficients of very high resolution.
(Otherwise phase extension would be impossible)  This problem was discussed in 
detail in Lynn Ten Eyck's 1976 paper on Fcalc FFT's but is often 

[ccp4bb] AW: [ccp4bb] double cell dimensions between P2 and C2

[Herman . Schreuder]

Dear Mark,

It does happen, even that two crystals from the same drop (everything 
identical) have different space groups.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Markus 
Heckmann
Gesendet: Donnerstag, 9. November 2017 11:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] double cell dimensions between P2 and C2

Dear all,
From a small protein, gives crystals P2 with cell
Cell 53.16   65.73   72.8990  110.94  90
(has 3 molecules in the asymmetric unit). Tested with pointless. Does not give 
any other possibility.

Another crystal if the same protein, similar conditions:
C2
Cell 109.14  124.37   73.4290  111.75  90. This has 6
molecules in the a.s.u. Tested with pointless. Does not give any other 
possibility.
The cell length a, b of C2 is twice that of P2.

Is it usual to get such crystals from similar conditions or am I missing 
something?

Many thanks,
Mark

[ccp4bb] AW: Radiation damage to the FAD in enzyme structure

[Herman . Schreuder]

Dear Martin,

You could calculate an Fo-Fc map with the FAD having half occupancy. This 
should bring out the "pure" difference density for your modified FAD, which 
might be easier to interpret. You may have to try different occupancies, say 
0.4, 0.45, 0.5, 0.55, 0.6 etc. to find the point with the least residual intact 
FAD density.

In the absence of any pictures of the electron density and any information 
about which ring is almost missing and how the other rings are distorted, it is 
very difficult, if not impossible to provide you with any clues.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Martin 
Malý
Gesendet: Dienstag, 7. November 2017 17:00
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Radiation damage to the FAD in enzyme structure


Dear colleagues,

thanks for all your responses. Just to make things clear, we are absolutely 
sure about 100% presence of the FAD in the original solution. What we see in 
the electron density is roughly half occupancy for the FAD and additional 
difference density for something what looks like a product of some FAD 
degradation. Either caused by degradation or age. The product looks like one of 
the three planar ring is almost missing, the other two are significantly 
distorted. We see it clearly, but we want to honestly and correctly interpret 
the density based on some prior chemical knowledge. Does anyone has a clue?

In the mean time, we are reading the other literature provided by you, of 
course.


Best regards,

Martin


Od: CCP4 bulletin board > 
za uživatele Martin Malý >
Odesláno: 6. listopadu 2017 12:01:43
Komu: CCP4BB@JISCMAIL.AC.UK
Předmět: [ccp4bb] Radiation damage to the FAD in enzyme structure

Dear colleagues,

I am investigating a structure of a FAD-dependent enzyme. The electron density 
map suggests radiation damage to the FAD. It apparently is different from 
simple change of the redox state and "butterfly"-like structure. We did not 
find in literature possible products of radiation damage, like a removal of 
several atoms of the FAD. Has anyone observed such effect?

To describe it in more detail, I can observe negative difference map of C2, N3, 
C4, and O4 atoms of flavine. Moreover, there is positive difference map close 
to O2 and O4 atoms thus it looks as water molecules are bound there instead of 
the missing FAD atoms.

I am attaching parameters of the experiment: performed at synchrotron,
exposition time 210 s, high resolution diffraction limit 1.65 A
( = 2 at shell 1.75-1.65 A). We could see a decrease of
diffraction data statistics during the experiment hence we think there
is significant radiation damage to the crystal.

Thank you very much for ideas.
Regards,
Martin Maly

[ccp4bb] AW: Re: [ccp4bb] Removing a ter line present in the middle of the chain

[Herman . Schreuder]

My feeling is that all non-standard amino acids should be labeled HETATM and 
that the overzealous introduction of TER cards by Refmac is the bad thing, but 
I might be mistaken…
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Dienstag, 7. November 2017 13:28
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Removing a ter line present in the middle of 
the chain

Well - I just edited the HETATM to ATOM  and the TER went away..
Something (Refmac? Coot?? mystery?) was labelling all atoms in MSE residues as 
HETATM and that was a BAD THING..
Eleanor


On 7 November 2017 at 08:05, Abhishek Anan 
> wrote:
Are you LINK records properly defined? You might want to check them. I
had a similar issue and updating links fixed it.

Best
Abhishek

On 11/7/17, Eleanor Dodson 
> wrote:
> No - it is not.
>
> I have seen similar problems.
> Your atom records arent labelled as HETATM are they?
>
> That triggers strange behavior.
> Eleanor
>
>
> On 7 November 2017 at 07:13, Abhishek Anan 
> >
> wrote:
>
>> Are you adding the cif file of the unnatural amino acid on LIB in path
>> in refmac.
>>
>> Best,
>> Abhishek
>>
>>
>>
>> On 11/7/17, Rashi Aggarwal 
>> > wrote:
>> > Dear all,
>> >
>> > I have an unnatural amino acid in my structure which I could
>> > successfully
>> > add in coot. The amino acid is taking the right bonds when viewed with
>> > coot. However, the pdb file has a ter line just above the residue.
>> >
>> > If I remove this line and submit the pdb to refmac it again adds the
>> > ter
>> > line, I can still remove it and go ahead with validation but is it the
>> > right thing to do?
>> >
>> > Thanks
>> >
>> > Best,
>> > Rashi
>> >
>>
>

[ccp4bb] AW: Radiation damage to the FAD in enzyme structure

[Herman . Schreuder]

Dear Martin,

For well-refined structures with low residuals, +/- 3 sigma levels (the 
standard coot contour levels) are very small on an absolute scale. How does 
your 2Fo-Fc map look like? Are there really big holes in the FAD for the 
missing atoms with almost zero 2Fo-Fc density?

As others have mentioned, the most likely explanation will be that your 
ringsystem lost some planarity, which may be a butterfly motion, but also a 
twisting. I would edit your FAD cif file and reduce the planarity restraints at 
least with a factor of 10 and better even more, do a real space refinement and 
see what happens. For testing purposes, you could also remove the planarity 
restraints completely. At 1.65 Å you may have to move the atoms manually into 
the positive difference density since they otherwise may remain stuck in the 
nearby false minimum.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Martin 
Malý
Gesendet: Montag, 6. November 2017 12:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Radiation damage to the FAD in enzyme structure

Dear colleagues,

I am investigating a structure of a FAD-dependent enzyme. The electron density 
map suggests radiation damage to the FAD. It apparently is different from 
simple change of the redox state and "butterfly"-like structure. We did not 
find in literature possible products of radiation damage, like a removal of 
several atoms of the FAD. Has anyone observed such effect?

To describe it in more detail, I can observe negative difference map of C2, N3, 
C4, and O4 atoms of flavine. Moreover, there is positive difference map close 
to O2 and O4 atoms thus it looks as water molecules are bound there instead of 
the missing FAD atoms.

I am attaching parameters of the experiment: performed at synchrotron,
exposition time 210 s, high resolution diffraction limit 1.65 A
( = 2 at shell 1.75-1.65 A). We could see a decrease of
diffraction data statistics during the experiment hence we think there
is significant radiation damage to the crystal.

Thank you very much for ideas.
Regards,
Martin Maly

[ccp4bb] AW: Re: [ccp4bb] another unknown density problem

[Herman . Schreuder]

You are right. In this case, I would put some waters in it, refine and see if 
the density gets any clearer. However, since from this perspective the density 
is quite far away from the protein, it could be a very disordered PEG, which, 
even at high resolution, might be impossible to fit.

Best,
Herman

Von: Abhishek Anan [mailto:rendezvous.a...@gmail.com]
Gesendet: Freitag, 3. November 2017 11:10
An: Schreuder, Herman /DE
Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] another unknown density problem

Dear Prof Schreuder
Here are another couple of perspectives from coot. The density is too far and 
isolated from the peptide chain to be an alternate conformation or 
conformational change. The density of the peptide chain does not look good 
because it was truncated at 5A for clarity.
Best regards
Abhishek

On Fri, Nov 3, 2017 at 9:33 AM, 
> wrote:
Dear Abhishek,

To me, it looks like an alternative conformation of the peptide chain or maybe 
even a conformational change with respect to the starting model. The peptide 
chain does not look too well defined, despite high resolution electron density.

Best,
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Abhishek Anan
Gesendet: Freitag, 3. November 2017 09:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] another unknown density problem

Hi all,
I have an "unknown" density in the map. I have tried to fit it to PEG but it 
doesn't fit very well. I was wondering if there are other PEG-related or other 
molecules I could try.
The crystal grew in TRIS-HCl and PEG MME 2K.
Thank you
Abhishek

[ccp4bb] AW: [ccp4bb] another unknown density problem

[Herman . Schreuder]

Dear Abhishek,

To me, it looks like an alternative conformation of the peptide chain or maybe 
even a conformational change with respect to the starting model. The peptide 
chain does not look too well defined, despite high resolution electron density.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Abhishek 
Anan
Gesendet: Freitag, 3. November 2017 09:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] another unknown density problem

Hi all,
I have an "unknown" density in the map. I have tried to fit it to PEG but it 
doesn't fit very well. I was wondering if there are other PEG-related or other 
molecules I could try.
The crystal grew in TRIS-HCl and PEG MME 2K.
Thank you
Abhishek

[ccp4bb] AW: Re: [ccp4bb] TYC not linked to previoys amino-acid

[Herman . Schreuder]

Dear Eleanor,
The method I proposed works for any amino acid, not just tyrosine. Having an 
amide as a separate residue may sound strange, but is it exactly how our 
chemists treat it as well.
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Montag, 16. Oktober 2017 13:40
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] TYC not linked to previoys amino-acid

Thank you Garib..


So you have to copy the $CLIBD/monomers/t/TYC.cif to your own directory and 
change the string "non-polymer" to "L-peptide"
Then use that as a dictionary entry and indeed that behaves as expected - ie 
forms the peptide link GLY-TYC
Eleanor

On 16 October 2017 at 12:32, Garib Murshudov 
> wrote:
These are default values.

Decision about standard residue types are made using the info from the 
dictionary.  In the current version it is like:

TYC  TYC 'L-TYROSINAMIDE  ' non-polymer25  13 .

I.e. it is not a peptide. Strictly speaking it is not a peptide. It would form 
standard peptide link, at least on the carboxyl side.
These cases should be dealt with different types of links.

How this residue incorporated into the protein? Is it something like C-terminus 
amilation? If it is the case then better approach is to use TYR and C-terminus 
amilation modification. I do not remember how it works, but if it is the case 
then i can find out.


Regards
Garib


On 16 Oct 2017, at 07:06, Eleanor Dodson 
> wrote:


Very puzzled by this - but now I think I understand .
Can you comment Garib please?
In refmac there is a list of amino acids to be linked

  SUBROUTINE GET_STANDARD_RES_TYPE(MDOC,LINE,MONN,ITYPE,IERR)
 ...
C -
C ITYPE = 3 RES_LTYPE( 3) =  'peptide '
C
  DATA RNAME /'CYS','SER','THR','PRO','ALA','GLY','ASN','ASP'
 *   ,'GLU','GLN','HIS','ARG','LYS','MET','ILE','LEU'
 *   ,'VAL','PHE','TYR','TRP','TRY','HYP','PCA','SAR'
 *   ,'ORN','CSS','CSH','DAR','DPR','NLE','DAS','INI'
 *   ,'DGL','DGN','DHI','DIL','DIV','DLI','DSN','DSP'
 *   ,'ABA','BMT','MLE','MVA','DVA','MSE','PTR','DAL'
 *   ,'DPN','DTR','DTH','TYS','CGU','DCY','ILG','OCS'
 *   ,'KCX','SAH','SAM','SEP','LLP','5HP','CSO'  /



As you can see TYC is not in this list:

When I change your TYC to TYS the link is formed properly,  ( I have to delete 
the NXT but that shouldnt matter..)

So either TYC and all other possible peptides should be added to the RNAME 
list, or better if the dictionary defines a residue as an L-peptide then that 
is how it should be handled.


But for the present you will have to use that work-around of creating a LINK.

Eleanor
C


Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge
CB2 0QH UK
Web 
http://www.mrc-lmb.cam.ac.uk,
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

[ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[Herman . Schreuder]

Dear Michael,

Did you ask Phaser to check for all possible space groups? There are still I422 
and I4 you did not mention. If the space group that came out of Phaser is 
different from the space group used for processing, subsequent refinement 
programs may use the wrong space group from the processing. This should be easy 
to check.

The other suggestion I have is to try a different processing program. Although 
XDS is excellent, I find that sometimes it has difficulties with ice rings, 
which reveal themselves not in the processing, but in the subsequent 
refinement. You may want to try Mosflm or some other processing program.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Michael 
Jarva
Gesendet: Sonntag, 15. Oktober 2017 03:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Another troublesome dataset (High Rfree after MR)

To add to the current anisotropic discussion I recently got a dataset I'm 
unable to refine and I'm hoping I could get some help on figuring out if 
there's anything I can do.

I get a clear cut solution with Phaser using the same protein as search model 
and got a TFZ of >16, LLG >200, and a packing that makes sense, so I don't 
doubt the solution. However, the maps look terrible, more like something I 
would expect from a 3.65Å dataset rather than the 2.65Å it supposedly is.

The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 5% and a 
CC1/2 of >0.5 in the outer shell (see the bottom for full summary). There is 
some minor radiation damage but I could cut out most of it due to the high 
symmetry.

Xtriage reports no indication of twinning, but does say that the data is 
moderately anisotropic, so I ran the unmerged data through the StarAniso 
server, which reported the ellipsoidal resolution limits to be 2.304, 2.893, 
and 3.039. Refining with the anisotropically truncated data improves the maps 
somewhat, but I am still unable to get the Rfree below 38%. I tried using both 
phenix.refine and buster with similar results.

I've considered the choice of space group and tried I41, F222, I212121 , and 
C2, but with the same results, and Zanuda tells me the same thing.
Lastly, there is some minor ice rings, so my last try was to exclud the ice 
ring resolutions, but this made little to no difference.

Normally I would just write this off as the data being bad but this time all 
the statistics tell me this should be doable so I'm curious what has gone wrong.

Cheers
Michael Jarva



Summary data forProject: XDSproject Crystal: XDScrystal Dataset: 
XDSdataset



   Overall  InnerShell  OuterShell

Low resolution limit   34.87 34.87  2.78

High resolution limit   2.65  8.79  2.65



Rmerge  (within I+/I-) 0.052 0.026 1.595

Rmerge  (all I+ and I-)0.057 0.030 1.805

Rmeas (within I+/I-)   0.062 0.031 1.924

Rmeas (all I+ & I-)0.063 0.033 1.993

Rpim (within I+/I-)0.032 0.017 1.042

Rpim (all I+ & I-) 0.025 0.014 0.817

Rmerge in top intensity bin0.030- -

Total number of observations   19931   566  2681

Total number unique 3597   114   471

Mean((I)/sd(I)) 11.3  42.3   0.8

Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.575

Completeness97.9  93.1  99.6

Multiplicity 5.5   5.0   5.7



Anomalous completeness  92.4  92.1  96.8

Anomalous multiplicity   3.0   3.0   3.0

DelAnom correlation between half-sets  0.176 0.258 0.051

Mid-Slope of Anom Normal Probability   1.078   - -



Average unit cell:   82.39   82.39   69.73   90.00   90.00   90.00

Space group: I 41 2 2

Average mosaicity:   0.10

[ccp4bb] AW: [ccp4bb] High R/Rfree after MR

[Herman . Schreuder]

Dear GIA,

In addition to the anisotropy, I would also check your diffraction images and 
make sure that there are no (even hardly perceptible) ice rings present. 
Depending on how the data processing software handled this, they may cause high 
Rfactors in the range you mentioned.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Gianluca 
Cioci
Gesendet: Freitag, 13. Oktober 2017 10:30
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] High R/Rfree after MR

Dear All,

I am trying to refine a structure at 3.3A. Model has 60% identity to the 
target. Maps look OK (for 3.3A) and rebuilding in Coot is relatively 
straightforward. However, after some rebuilding cycles the R factors are stuck 
at 0.37/0.39 (REFMAC).
XTRIAGE tells me that everything is normal (no twin, 98% completeness, R=3.5% 
in the low resolution bin), perhaps some anisotropy is present.
I have already refined 2 homologous structures at resolutions going from 3.2 to 
3.8 and there were no problems (final R ~ 0.21/0.24).
Any advice ?

Thanks,

GIA

[ccp4bb] AW: [ccp4bb] C-terminal amide

[Herman . Schreuder]

Hi Abhisek (and BB),

I use the attached cif file. It has an NH2 residue defined as a peptide and 
gets automatically linked to the peptide chain in the buster procedure I use. 
So if your last residue is Tyr 100, you add NH2 101 as a HETATM in the peptide 
chain.

I have not tested it with Refmac though.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Bernhard 
Lechtenberg
Gesendet: Freitag, 13. Oktober 2017 00:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] C-terminal amide

HI Abhisek,

I had the same problem a few years back. Here is the solution I came up with 
thanks to help from the CCP4bb:

1) Add pointer atom in Coot NH2 and create link to C-terminal residue (Coot: 
Extensions -> Modeling -> Make link)
2) create cif file with correct link description (see below)
3) modify LINK record in PDB file to correct residues/ link name (TYR-NH2 if 
you use the file below with modifications for your case)
4) refine in Refmac with cif as LIB in

Augie Pioszak sent me the cif file copied below. you will have to change PHE to 
TYR for your particular case.

Hope that helps.

Best,
Bernhard

---

Bernhard,
You need a link record in the pdb file to link the NH2 amino group to your last 
peptide residue.  When I did this a few years back I had to include a .cif 
library file describing the link for use with Refmac.  See pdb entry 3c4m and 
below for the lib description I used.  I still use O, so not sure how coot 
handles it, but I would guess it will recognize the NH2 group just fine.  Hope 
this helps.
Regards,
Augie Pioszak

# ---   LIST OF LINKS ---
#
data_link_list
loop_
_chem_link.id
_chem_link.comp_id_1
_chem_link.mod_id_1
_chem_link.group_comp_1
_chem_link.comp_id_2
_chem_link.mod_id_2
_chem_link.group_comp_2
_chem_link.name
PHE-NH2  PHE  ..NH2  ..
bond_PHE-C_=_NH2-N
# --
#
# --- DESCRIPTION OF LINKS ---
#
data_link_PHE-NH2
#
loop_
_chem_link_bond.link_id
_chem_link_bond.atom_1_comp_id
_chem_link_bond.atom_id_1
_chem_link_bond.atom_2_comp_id
_chem_link_bond.atom_id_2
_chem_link_bond.type
_chem_link_bond.value_dist
_chem_link_bond.value_dist_esd
PHE-NH2  1 C   2 N single  1.3290.020
loop_
_chem_link_angle.link_id
_chem_link_angle.atom_1_comp_id
_chem_link_angle.atom_id_1
_chem_link_angle.atom_2_comp_id
_chem_link_angle.atom_id_2
_chem_link_angle.atom_3_comp_id
_chem_link_angle.atom_id_3
_chem_link_angle.value_angle
_chem_link_angle.value_angle_esd
PHE-NH2  1 O   1 C   2 N   122.0001.600
PHE-NH2  1 CA  1 C   2 N   118.2002.000
loop_
_chem_link_plane.link_id
_chem_link_plane.plane_id
_chem_link_plane.atom_comp_id
_chem_link_plane.atom_id
_chem_link_plane.dist_esd
PHE-NH2plane11 CA0.020
PHE-NH2plane11 C 0.020
PHE-NH2plane11 O 0.020
PHE-NH2plane12 N 0.020
# --

-
> On Oct 12, 2017, at 2:53 PM, Abhishek Anan  wrote:
> 
> Dear all,
> 
> I am trying to solve the structure of a peptide with C-terminal amide. In 
> order to add the amide group to the c-terminal TYR, I substituted TYR with 
> TYC (tyrosinamide) and created a link between the preceding GLY and TYC. When 
> refined in refmac, the pdb inserts a TER card between GLY and TYC and no 
> covalent bond is created between them. How do I fix this? I have also tried 
> to add NH2 pointer atom to TYR in coot and create a link between them but in 
> vain. I also imported the NH2.cif file into coot and tried to make a link but 
> of no use. I also tried to import NH2.cif into Jligand so I could get a link 
> record but it refuses to open the NH2.cif file. 
> 
> I would greatly appreciate any help!
> 
> Best regards,
> Abhishek


NH2.cif
Description: NH2.cif

[ccp4bb] AW: [ccp4bb] include corners in mosflm

[Herman . Schreuder]

With a detector in swing-out position, one has to include the corners. Also, 
why should one discard potential data during processing? Based on the 
statistics, one can always discard data afterwards if it is not good or too 
incomplete.

HS

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Keller, 
Jacob
Gesendet: Dienstag, 26. September 2017 22:14
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] include corners in mosflm

Why on earth would one want that to be the *default*? I understand that there 
may be the odd unrepeatable dataset collected too close, or there may be 
occasionally be hardward limitations, but I cannot understand how this would be 
a recurring problem….

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CCP4BB
Sent: Tuesday, September 26, 2017 4:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] include corners in mosflm

Hi Ed

I'm afraid not; that's one thing that can't be changed to a different default.
Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing)

On 26 Sep 2017, at 20:34, Edwin Pozharski 
> wrote:
By default, iMosflm excludes corners from processing.  Is there a simple way to 
make it the default to go all the way to the corner instead of detector edge?  
I could of course set the max resolution for processing to some outrageous 
value that is guaranteed to be outside of the image, but perhaps I am missing a 
more intelligent option in the gui.  (I vaguely recall HKL2000 having a 
Edge/Corner/Other) radiobutton).

There is a whole separate question as to wisdom of including corners, of 
course.  Yes, adding a resolution shell with robust data will improve model 
quality even if such shell is woefully incomplete. On the other hand, it's 
possible that fill-in option for missing reflections in map calculation may 
make maps more biased. A reasonable solution to this would be to use 2 
different resolution limits in refinement and map calculation - not hard to 
script for that yet I don't know if any refinement software provides such 
option natively.
Ed.

[ccp4bb] AW: [ccp4bb] Precipitation issue during refolding

[Herman . Schreuder]

Hi Dipankar,

Are you expressing the active protease, or the inactive precursor (zymogen)? 
Although we never systematically looked at it, I strongly suspect that many 
active proteases destroy the expression host and you will therefor only find 
clones expressing the protein in inclusion bodies.

Upon refolding, autoproteolysis may become a problem. Since it is a bimolecular 
process, the rate goes up with the square of the concentration. So during the 
concentration steps, you may have to add a strong inhibitor of your protease. 
If it is a new structure, you could consider adding an irreversible, covalent 
inhibitor to completely inhibit your protease. PPACK has been used for this in 
a large number of proteases, but it depends on the substrate specificity of 
your protease. Alternative, you could mutate the active site serine into an 
alanine.

So in summary:
-produce the protease as a zymogen and activate afterwards. There is a reason 
nature produces most proteases as zymogens.
-make sure you prevent autolysis during refolding and especially during 
concentration.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dipankar 
Manna
Gesendet: Donnerstag, 21. September 2017 12:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Precipitation issue during refolding

Hi,

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with.
​
Any suggestion to deal this issue would be highly appreciated.

Thank you in advance.

Best,

Dipankar​

--
Dipankar Manna, Ph.D
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517
E-mail: dipankar.ma...@medisin.uio.no
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/

[ccp4bb] AW: [ccp4bb] doubt regarding MR search model

[Herman . Schreuder]

Dear Satvik,

An R/Rfree of 0.29/0.35 after one round of automatic model building indicates 
that your solution is correct. You can proceed with refinement and rebuilding. 
You can take either the monomer-based or dimer-based solution, it does not 
matter. Personally I would take the monomer-based solution since it allowed for 
some small difference in orientation of the monomers within the dimer.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Satvik 
Kumar
Gesendet: Mittwoch, 20. September 2017 14:40
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] doubt regarding MR search model

Hello,
Thanks all for your valuable suggestions. Two things that stuck me were "one 
gets to know if the space group is correct only after solving the structure" 
and "to try all possible space groups until one lands up with the correct 
solution".
I ran Phaser with "all alternative space groups". The solution is still in the 
same space group P212121. This holds true with both the monomer as well as 
dimer search models. I hope I have checked all orthorhombic space groups by 
doing this as I am unsure what "all alternative space groups" means.
I also ran Zanuda which tells me that the space group P212121 is correct in 
both cases.
Pointless output indicates that "Systematic absence probability: 0.818". Can I 
take this to be convincing or do I need to run other tests?

I dont know how to check for non-crystallographic symmetry? Please tell me how 
I can do so.

The model is 60 percent identical to my protein. Also, one round of automated 
model building has brought down the R values. The Rwork,Rfree stand at 
0.29,0.35 (monomer search model) and 0.30, 0.35 (dimer search model). There is 
good scope for building residues in both cases. But which one do I go ahead 
with is my question.


Thanks,
Satvik


On Tue, Sep 19, 2017 at 11:40 PM, Eleanor Dodson 
> wrote:
Well - you haven't said what the sequence identity between model and your 
protein is, nor if you have a non-crystallographic translation.

With low homology that R factor drop is acceptable and rebuilding can fix it, 
However if there is high homology you might expect better.

But this sort of conjecture is pretty pointless - check in all orthorhombic  
space groups as Mark suggests.

Eleanor

On 19 September 2017 at 15:16, Mark J van Raaij 
> wrote:
With Rs of 43/48% I don't think you can be sure that your spacegroup is right.
You should always try all the spacegroup possibilities until you get a solution 
you are sure is right, i.e. that refines to Rs of around 35% or preferably even 
lower.
More so in the case of screw axes, so try P222, P2122, P2212, P2221, P21212, 
P21221, P22121 and P212121. Phaser can do this automatically for you by 
clicking the right box.
If necessary, then try lower symmetry like P21 and perhaps P1.
Programs like Xanuda can help.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 
3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/

On 19 Sep 2017, at 16:01, Satvik Kumar 
> wrote:

Hello,
Thanks everyone for your explanations.
I have pasted the pointless output to provide more information.

Best Solution:  space group P 21 21 21

Laue group probability:   0.959

Systematic absence probability: 0.818

Total probability: 0.785

Space group confidence:0.751

Laue group confidence   0.951


Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00

Also based on L-test, pointless says data does not suggest twinning.

Yes, the R values go down when I refine in both cases. After 20 rounds of 
restrained refinement using the coordinates generated by monomer as search 
model, the Rwork and Rfree are 

[ccp4bb] AW: [ccp4bb] His-6 versus His-10 tag

[Herman . Schreuder]

Dear everybody who reacted,

It seems that when the His6 construct crystallizes, there is a fair chance that 
the his10 construct will crystallize as well. However, as usual in 
crystallography there is no guarantee, so I have added a TEV site to the 
construct in order to be able to remove the tag if necessary.

Thank you all very much!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Savvas 
Savvides
Gesendet: Dienstag, 19. September 2017 16:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] His-6 versus His-10 tag

Hi Herman
the survey article by Carson in Acta D
DOI:

10.1107/S0907444906052024
from a decade ago may provide relevant information/insights.
best wishes
Savvas




On 19 Sep 2017, at 12:10, 
herman.schreu...@sanofi.com wrote:

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman

[ccp4bb] AW: [ccp4bb] doubt regarding MR search model

[Herman . Schreuder]

Dear Satvik,

You only know if the space group is correct AFTER you solved your structure. 
With an Rwork/Rfree of 0.43/0.48, the space group is likely not correct. The 
way to solve this is to run MR in all possible space groups. Most, if not all 
MR programs have an option to do this automatically.

For phaser e.g. you have to give the command: SGALTERNATIVE SELECT ALL
If your search model is not too different from the crystallized protein, this 
should work without a problem. Otherwise, you get into the realm of difficult 
molecular replacement and that will be a different story.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Satvik 
Kumar
Gesendet: Dienstag, 19. September 2017 16:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] doubt regarding MR search model

Hello,
Thanks everyone for your explanations.
I have pasted the pointless output to provide more information.

Best Solution:  space group P 21 21 21

Laue group probability:   0.959

Systematic absence probability: 0.818

Total probability: 0.785

Space group confidence:0.751

Laue group confidence   0.951


Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00

Also based on L-test, pointless says data does not suggest twinning.

Yes, the R values go down when I refine in both cases. After 20 rounds of 
restrained refinement using the coordinates generated by monomer as search 
model, the Rwork and Rfree are 0.43 and 0.48
respectively. Refinement using the coordinates generated by using dimer as 
search model also results in similar R values. I have attached the plots to 
show that the R values indeed reduce in both cases.
Is my space group correct? Do I need to reexamine the space group even though 
the probability is high?

If my space group is indeed correct, how do I decide whether to go ahead with 
the results generated by the monomer search model or the dimer?
Please share your thoughts.

Thanks,
Satvik

On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson 
> wrote:
You need to provide a bit more information.
First of all about the data processing..
Is the space group correct?
ways of being misled are:
Non-crystallographic translations with a shift of ~0.5 along an axis - say a.  
This will generate absences in the odd h 0 0 reflections and can make the space 
group appear to be P 21 21 21 whilst it is really P 2 21 21..
Perfect twinning can have the same effect. In an orthorhombix space group this 
can usually only occur if two axes have approximately the same length, but the 
data processing stats can indicate if that is the case.
Then - re PHASER. The packing rejection criteria may be set too severely - that 
seems the case for your solution.
Best check on any MR solution is: does it refine - give it 20 cycles of 
mindless refinement and see if the R and FreeR go down.
Then look at the maps and see if there are obvious corrections to be made..
Eleanor

On 18 September 2017 at 14:59, Satvik Kumar 
> wrote:
Dear Crystallographers,
I am trying to solve a structure in the space group P212121. Based on Matthews 
coefficient, there are 4 molecules in the asymmetric unit.
Based on my limited reading about using of Phaser, I understand that a single 
chain should be used as search model even though many copies are present in 
asymmetric unit. Am I correct?
So when I use a single chain as search model and ask Phaser to search for 4 
molecules, Phaser identifies a single solution with a warning "The top solution 
from a TF rescoring did not pack" and a warning "Search request requires more 
scattering than defined in composition. Composition increased to accommodate 
search components". But the final values reported "PAK=2 LLG=1065 TFZ==22.6" 
indicate that phaser has solved the problem.

Can anyone please explain the meaning of the warning.
When I inspect the arrangement of the chains (attachment), I observe minimal 
contact between the chains and a large cavity in the center. Can a crystal form 
this way?

I have also tried using the dimer as search model and asking phaser to search 
for 2 molecules. Even in this case, Phaser finds a single solution but the 
warning and the advisory still appear as before. The numbers reported reduce a 
bit to "PAK=1 LLG=722 TFZ==29.2".
Please help me in understanding these results.
Thanks,
Satvik

[ccp4bb] AW: [ccp4bb] question regarding sequence numbering

[Herman . Schreuder]

Hi Dave and Tony,

Upon submission, the pdb checks the sequence and automatically generates 
comments about sequences derived from the expression vector. So you do not have 
to do anything. Given the issues many programs have with non-sequentially 
numbered residues, I would also number them 7,8,9.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Briggs, 
David C
Gesendet: Dienstag, 19. September 2017 14:24
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] question regarding sequence numbering

Hi Tony,
When I've had similar issues, I've numbered them sequentially (i.e.  7,8,9) and 
remarked in the PDB header that they are vector-derived sequence.
I believe that is what the PDB ask you to do in situations like this (maybe 
they can comment?).
If they are not numbered sequentially, then often graphics and refinement 
software won't treat them as linked.
Dave
--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: Antonio Ariza
Sent: Tuesday 19 September, 13:15
Subject: [ccp4bb] question regarding sequence numbering
To: ccp4bb@jiscmail.ac.uk

Hi all,
Here's a problem I haven't come across before. I'm working on a structure whose 
expression plasmid was designed to remove the first 9 amino acids from the 
protein of interest and to which an N-terminal tag was added. After cleaving 
the tag I am left with 3 amino acids (GPM) followed by the original sequence. 
Obviously the residues of interest should follow the numbering of the original 
sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?
Cheers,
Tony
--
Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
Structural insights into the function of 

 
ZRANB3
 in replication stress 
response

[ccp4bb] His-6 versus His-10 tag

[Herman . Schreuder]

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman

[ccp4bb] AW: [ccp4bb] A question about Cys and fluoro benzene ring

[Herman . Schreuder]

Dear Cheng,

You could check whether you get irreversible inhibition with your compound, 
which could be a sign of a covalent link with the protein.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Savvas 
Savvides
Gesendet: Mittwoch, 23. August 2017 21:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] A question about Cys and fluoro benzene ring

Dear Cheng,

it is possible that the sulfhydryl of the cysteine might have reacted with the 
methyl fluoro benzyl group via nucleophilic aromatic substitution. Fluorine is 
not the best leaving group among the halides but it would certainly allow this 
especially if the ligand-protein incubation time was sufficient and if the the 
pH of the reaction is above 7. This would get the Cys-SH closer to the 
cysteinyl moiety (S:-). The pKa of the Cys-SH group is just under 8.5 and in 
your case it might even be lower depending on local structure environment, 
resulting in an even better nucleophile.

Your electron density indeed suggests that the sulfur from the Cys-SH is 
covalently bonded to the benzyl ring and this would happen at the carbon that 
originally carried the fluorine. You will have to remodel your ligand, keeping 
in mind that the fluorine will no longer be part of your ligand model. The 
methyl group will of course be retained.

It might also be useful to try to confirm the reactivity of the ligand of 
interest with the protein (reminiscent of a suicide or covalent inhibitor 
against cysteine proteases?) via some kind of an orthogonal method (e.g. mass 
spectrometry). This would even tell you if the particular cysteine in your 
structure is truly the relevant covalent target. In this way you can even 
compare washed and dissolved crystals of your protein-ligand complex with the 
complex in solution.

best wishes
Savvas


---
Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx




On 23 Aug 2017, at 17:01, Cheng Zhang 
> wrote:

Hi everyone,

We recently got a structure of a receptor bound to a ligand. The ligand has a 
fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
receptor. The density for the ligand and the Cys seems to suggest a covalent 
bond. However, I don't know if a covalent bond is chemically possible. Also, I 
believe Cys is rarely involved in cation-pi interactions? Any suggestions for 
placing the Cys and the fluoro methyl benzene ring?

Thanks!

Cheng



--
-
Cheng Zhang

[ccp4bb] AW: [ccp4bb] Unknown positive electron density

[Herman . Schreuder]

Dear Betty,

You have very high resolution, which helps you to identify your ligand, but the 
ligand may be disordered…
What I would do is to place some dummy atoms (e.g. waters) and refine and look 
if the molecule gets clearer. By scrolling the density in coot, you can 
identify the positions with the highest electron density were you should put 
your dummy atoms.

Looking at your pictures, as far as is possible without the ability to rotate 
them, I would try to fit deoxyribose-phosphate, or even a complete nucleotide. 
Maybe your prep contained some unreacted nucleotides and your anomalous peak is 
phosphorus.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Betty Chu
Gesendet: Montag, 21. August 2017 17:19
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Unknown positive electron density

Dear ccp4bb,
I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.
Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,
Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park

[ccp4bb] AW: AW: [ccp4bb] high Rfree

[Herman . Schreuder]

Hi ???,

I would first try AMPLE, as was suggested by Daniel.

If separate helices still give you an anti-parallel coiled coil, your structure 
may in fact be anti-parallel. Your problem is not that the structure has 
another parallelity than you would like, but that you cannot solve the MR 
problem. If your structure turns out to be anti-parallel, that’s what it is, as 
long as the structure has been solved correctly.

How do your electron density maps look like: do you see any difference density 
for mutated side chains? Do you see any hints how to extend your shorter model? 
Did you do any refinement? If your MR solution is basically correct, the 
Rfactor might drop quite quickly during refinement, if not, the Rfactor may 
stay stuck or explode.

Best,
Herman


Von: 张士军 [mailto:21620150150...@stu.xmu.edu.cn]
Gesendet: Freitag, 21. Juli 2017 12:56
An: Schreuder, Herman /DE
Betreff: Re: AW: [ccp4bb] high Rfree


Hi

The resolution of my data is 2.8A, and the Rfree/Rwork are 0.55/0.49 which are 
very high. And I was separate them when I do MR, the solution is still  
anti-parallel coiled-coil after MR. Does it mean I MUST DO anomolous phasing ?
-原始邮件-
发件人:herman.schreu...@sanofi.com
发送时间:2017-07-21 15:37:33 (星期五)
收件人: 21620150150...@stu.xmu.edu.cn, 
CCP4BB@JISCMAIL.AC.UK
抄送:
主题: AW: [ccp4bb] high Rfree
Hi ???,

Coiled coil structures can be very tricky with MR, so your solution may not be 
correct. You could try to split your search model and run MR with the separate 
coils. If you have high enough resolution (> ~2.3 Å?) and your MR solution is 
basically correct, you may be able to solve your problem by rebuilding and 
refinement, but this may be a lot of work. Without any details (resolution of 
the data, current Rwork/Rfree etc.) it is difficult to give any more specific 
advice.

Best,
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Freitag, 21. Juli 2017 03:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short fragment 
recently, then I want to solve a longer fragment structure with phenix-MR using 
this short fragment structure as a model.The result is not good because of the 
Rwork and Rfree is 
high.So
 I think the longer fragment will be parallel coiled-coil which is different 
with the shorter one. I am wondering whether there are any other methods to 
handle this phenomenon besides heavy atom phasing? Thanks a lot!!!

[ccp4bb] AW: [ccp4bb] high Rfree

[Herman . Schreuder]

Hi ???,

Coiled coil structures can be very tricky with MR, so your solution may not be 
correct. You could try to split your search model and run MR with the separate 
coils. If you have high enough resolution (> ~2.3 Å?) and your MR solution is 
basically correct, you may be able to solve your problem by rebuilding and 
refinement, but this may be a lot of work. Without any details (resolution of 
the data, current Rwork/Rfree etc.) it is difficult to give any more specific 
advice.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Freitag, 21. Juli 2017 03:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short fragment 
recently, then I want to solve a longer fragment structure with phenix-MR using 
this short fragment structure as a model.The result is not good because of the 
Rwork and Rfree is 
high.So
 I think the longer fragment will be parallel coiled-coil which is different 
with the shorter one. I am wondering whether there are any other methods to 
handle this phenomenon besides heavy atom phasing? Thanks a lot!!!

[ccp4bb] AW: [ccp4bb] suggestions are welcome

[Herman . Schreuder]

Hi Gaoyina,

Maybe a stupid suggestion, but did the paper of the negative stain EM explain 
how the complex was prepared? That would be the first thing I would try.

Further, as Abbas explained, the complex may fall apart during gelfiltration. 
At least for analytical purposes, you could try to crosslink your dimer with 
glutardialdehyde and see how it behaves then during gelfiltration.

It could also be that you have to co-express both proteins to get the dimer.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Dienstag, 18. Juli 2017 04:25
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] suggestions are welcome

Hi all ,

It has been reported the Negative stain EM of a protein A-B complex, but 
according to my gel filtration results (I purified A and B respectively for 
incubation) , I found that A could not bind to B, of course I tried different 
buffer condition with various pH value, even the binding condition only had 50 
mm Kcl. Do you have any suggestion or methods that I can try to get the protein 
A-B complex?

Any suggestion is welcome，

Thank you all ,

Best,

[ccp4bb] AW: [ccp4bb] weird diffraction pattern

[Herman . Schreuder]

Hi Chenjun Tang,

From the images you sent, it looks like your crystal suffers from lattice 
translocation disorder. See e.g.
http://onlinelibrary.wiley.com/doi/10.1107/S0907444909025153/epdf

Calculating a native Patterson and looking for strange peaks may give some 
hints what is going on. Depending on the nature of the disorder, you may or may 
not correct for it.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tang 
Chenjun
Gesendet: Donnerstag, 13. Juli 2017 14:21
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] weird diffraction pattern

Hi David, 
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

[ccp4bb] AW: [ccp4bb] Rmergicide Through Programming

[Herman . Schreuder]

Dear All,

For me this whole discussion is an example of a large number of people barking 
at the wrong tree. The real issue is not whether data processing programs print 
amongst many quality indicators an Rmerge as well, but the fact that the PDB 
and many journals still insist on using the Rmerge as primary quality 
indicator. As long as this is true, novice scientist might be led to believe 
that Rmerge is the most important quality indicator. As soon as the PDB and the 
journals request some other indicator, this will be over. So that is where we 
should direct our efforts to.

I don't understand at all, why the PDB still insists on an obsolete quality 
indicator. However, the PDB format for the coordinates also dates back to the 
1960's to be used with punch cards.

My 2 cents.
Herman



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward 
A. Berry
Gesendet: Samstag, 8. Juli 2017 22:31
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Rmergicide Through Programming

But R-merge is not really narrower as a fraction of the mean value- it just 
gets smaller proportionantly as all the numbers get smaller:
RMSD of .0043 for R-meas multiplied by factor of 0.022/.027 gives 0.0035 which 
is the RMSD for Rmerge. The same was true in the previous example. You could 
multiply R-meas by .5 or .2 and get a sharper distribution yet! And that factor 
would be constant, where this only applies for super-low redundancy.

On 07/08/2017 03:23 PM, James Holton wrote:
>
> The expected distribution of Rmeas values is still wider than that of Rmerge 
> for data with I/sigma=30 and average multiplicity=2.0. Graph attached.
>
> I expect that anytime you incorporate more than one source of information you 
> run the risk of a noisier statistic because every source of information can 
> contain noise.  That is, Rmeas combines information about multiplicity with 
> the absolute deviates in the data to form a statistic that is more accurate 
> that Rmerge, but also (potentially) less precise.
>
> Perhaps that is what we are debating here?  Which is better? accuracy or 
> precision?  Personally, I prefer to know both.
>
> -James Holton
> MAD Scientist
>
> On 7/8/2017 11:02 AM, Frank von Delft wrote:
>>
>> It is quite easy to end up with low multiplicities in the low resolution 
>> shell, especially for low symmetry and fast-decaying crystals.
>>
>> It is this scenario where Rmerge (lowres) is more misleading than Reas.
>>
>> phx
>>
>>
>> On 08/07/2017 17:31, James Holton wrote:
>>> What does Rmeas tell us that Rmerge doesn't?  Given that we know the 
>>> multiplicity?
>>>
>>> -James Holton
>>> MAD Scientist
>>>
>>> On 7/8/2017 9:15 AM, Frank von Delft wrote:

 Anyway, back to reality:  does anybody still use R statistics to evaluate 
 anything other than /strong/ data?  Certainly I never look at it except 
 for the low-resolution bin (or strongest reflections). Specifically, a 
 "2%-dataset" in that bin is probably healthy, while a "9%-dataset" 
 probably Has Issues.

 In which case, back to Jacob's question:  what does Rmerge tell us that 
 Rmeas doesn't.

 phx




 On 08/07/2017 17:02, James Holton wrote:
> Sorry for the confusion.  I was going for brevity!  And failed.
>
> I know that the multiplicity correction is applied on a per-hkl basis in 
> the calculation of Rmeas.  However, the average multiplicity over the 
> whole calculation is most likely not an integer. Some hkls may be 
> observed twice while others only once, or perhaps 3-4 times in the same 
> scaling run.
>
> Allow me to do the error propagation properly.  Consider the scenario:
>
> Your outer resolution bin has a true I/sigma = 1.00 and average 
> multiplicity of 2.0. Let's say there are 100 hkl indices in this bin.  I 
> choose the "true" intensities of each hkl from an exponential (aka 
> Wilson) distribution. Further assume the background is high, so the error 
> in each observation after background subtraction may be taken from a 
> Gaussian distribution. Let's further choose the per-hkl multiplicity from 
> a Poisson distribution with expectation value 2.0, so 0 is possible, but 
> the long-term average multiplicity is 2.0. For R calculation, when 
> multiplicity of any given hkl is less than 2 it is skipped. What I end up 
> with after 120,000 trials is a distribution of values for each R factor.  
> See attached graph.
>
> What I hope is readily apparent is that the distribution of Rmerge 
> values is taller and sharper than that of the Rmeas values.  The most 
> likely Rmeas is 80% and that of Rmerge is 64.6%.  This is expected, of 
> course.  But what I hope to impress upon you is that the most likely 
> value is not generally the one that you will get! The distribution has a 
> width.  Specifically, Rmeas could be as 

[ccp4bb] AW: [ccp4bb] off-topic:fluorescence polarization displacement assay

[Herman . Schreuder]

Dear Megha,

I am puzzled by the results you presented. If you only see the effect in the 
presence of your protein, the protein must have something to do with it. The 
steep decline points to some highly cooperative effect, which might be 
aggregation/precipitation. Did you check that your protein is still in solution 
beyond the point of steep decline? Precipitation of your protein may explain 
what you are seeing.

My 2 cts,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von megha 
abbey
Gesendet: Mittwoch, 21. Juni 2017 21:21
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] off-topic:fluorescence polarization displacement assay

Hello All,

This is an off-topic question. I have some issues regarding Fluorescence 
Polarization competitive displacement assay and would need some advice.

I have developed an in vitro fluorescence polarization based assay using a 
N-terminus labelled FITC peptide. The peptide is 21 amino acids long and the 
binding protein is 50 Kda in size. The Kd for interaction is 1 uM. I am further 
running a competitive displacement assay using the exactly same unlabelled 
peptide. Here, with increasing concentration of the unlabeled peptide, the 
polarization signal first increases and then shows a sharp decline. Attached is 
the raw data file for the above.

I believe that if the unlabelled peptide had been aggregating, the polarization 
signal would increase but not drop. If the binding would have been 
non-specific, then the unlabelled peptide should not displace at all, but here 
I see an increase followed by a decrease in the signal. What does this increase 
and sharp drop in polarization signify and how do I fix this? Please help.

I have checked the polarization for titration of the unlabelled peptide mixed 
with fixed conc. of FITC-peptide (no protein added). Here, the polarization 
signals are the same for the entire range of unlabeled peptide. I have also 
tried incubating the unlabelled peptide with the protein (for ~15min) first 
followed by addition of FITC-peptide, but the results are the same.

Thank you,
Megha

[ccp4bb] AW: [ccp4bb] Unknown Ligand Density

[Herman . Schreuder]

Dear Nick,

I would contact some MassSpec people and ask if they could find out the mass of 
your mystery ligand. I would also carefully look at the neighboring protein. It 
might be an alternate conformation of a partially disordered loop. Finally, I 
would check literature to see if a natural ligand/cofactor of your protein 
would fit. However, your ligand seems to be bound in a crystal contact so it 
may be an artifact.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nick 
Thomas
Gesendet: Mittwoch, 14. Juni 2017 18:40
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Unknown Ligand Density

Dear CCP4bb,

I am refining a structure and have come across strong electron density for an 
unknown ligand (image attached). Would someone be able to identify this unknown 
ligand that somehow was co-purified with the protein.

The electron density shape does not match anything included in the 
crystallization conditions.

The crystallization condition is
0.1M MES monohydrate, pH 6.5, 1.6 M Magnesium sulphate heptahydrate

[ccp4bb] AW: [ccp4bb] help needed to interpret a SRF

[Herman . Schreuder]

Hi Vincent,

To calculate the best possible (self)rotation function, you want to have as 
many as possible intramolecular vectors (within the search molecule) and as 
little as possible intramolecular vectors (between molecules in the crystal). 
These latter vectors are meaningless (noise) as long as the orientation of your 
search molecule has not been found and you are not calculating a translation 
function.

Even with a small Patterson radius, you will get some intramolecular vectors 
near crystal contacts, but not many. With a large Patterson radius, you will 
get many intramolecular vectors which may obscure your rotation function 
solution.

So in difficult cases, you have to find the optimal radius: if it is too small, 
you won’t get much signal, if the radius is too large, you will get too much 
noise and you won’t see the correct solution either. The number of 
intramolecular vectors you get depend on the crystal packing and the shape of 
your molecule and there are no universally applicable hard rules. In your case 
it might be a good idea to run the (self)rotation function with different 
radii, too see which radius gives the best signal to noise ratio.

Hope this explains things a little,
Best, Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von vincent 
Chaptal
Gesendet: Freitag, 2. Juni 2017 12:05
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] help needed to interpret a SRF

Hi Michael,

thank you for your explanations.
On 01/06/2017 15:41, R. Michael Garavito wrote:
Vincent,

I see a few of problems with your SRF (the maps) which would impact the 
interpretation.

First you say that both crystals are processed as P21, which you would expect a 
very strong peak (100% of your origin peak) on kappa/chi=180 at the b* axis 
(one of the major axes of your map); this arises from the crystallographic 
symmetry.  Where this axis is depends on what the conventions are of the 
program you are using. Your SRF has a major peak along Z, but is that the b* 
axis?  (My b* axis is always placed North-South or your X, á la the Rossmann 
conventions.  I don’t know the conventions for Molrep) Then you see major 
off-axial peaks.  The sad thing about your off-axial peaks is that they are 
badly split.
The datasets were processed by XDS in primitive monoclinic P2 (space group 3), 
the reindexed to P21 by Aimless as the most probable space group. I just double 
checked that it is indeed P21, you made me doubt. I tried to look for the 
conventions for Molrep but couldn't find them, I'm sorry.
Following your 3rd comment bellow, I reproduced the maps with a radius of 30A 
as you suggested (see attached maps). I guess I can see the same features on 
both maps, with a peak along Y that would correspond to the 2 fold axis (if the 
convention is turned 90° compared to yours), and 2 off origin peaks along X. 
The maps from the crystal without additive is an awful mess. I should add that 
my monomer has an internal 2fold (pseudo)symmetry. Could the two main peaks 
mean that I have 2 monomers, each with his own 2 fold symmetry?

I'm puzzled by this radius of 30A as I read in the documentation that is 
corresponds to the approximate radius of the protein. My protein is about 
130Ax40A in the closed form to 130Ax70A in the open form according to other 
structures available. Shouldn't I compute maps with a radius of 70A or more? 
I'm not posting here these results here as they look more "artsy".

All the best
Vincent


The second problem is that the second crystal shows very strong peak (1001% of 
your origin peak) on kappa/chi=180, but at different position (along Y).  The 
control peak is the expected peak which arises from the P21 crystallographic 
symmetry.  If the data sets were processed as P21, they should be indexed with 
the 2-fold axis along b/b*, thus the control peak is the expected peak which 
arises from the P21 crystallographic symmetry.  Once that peak appears in the 
same place on each map, you can then compare the maps with more confidence.

The third problem is that all your maps have the “appearance" of mirror 
symmetry across the x-axis, which would be expected as it is the consequence of 
the P2(1) symmetry and you are looking at a hemisphere.  But it suggests that 
the 2-fold axis is along Y (b/b*). Then map 1 is missing the expected peak on 
kappa/chi=180 at the b* axis.

Final comment is that the maps labeling suggest that the radius of integration 
is 62-66 Å, which is way, way too large, even for an empty unit cell.  If it 
is, reduce it down to 20-30 Å to avoid intermolecular cross-peaks and see if 
the maps become clearer.  I have attached a SRF map from a P21 crystal form 
(radius of integration = 20 Å, resol. = 2.8 Å) with the 2-fold axis indexed 
along b/b* (Y) from polarrfn; note the “appearance" of mirror symmetry 
perpendicular to across the b/b*-axis (North-South or y-axis).

Cheers,

Michael




R. 

[ccp4bb] AW: [ccp4bb] Bond length and angle outlier fixing

[Herman . Schreuder]

Hi Vipul,

The first thing to do is to check whether the fit of the offending residues in 
the electron density maps is correct. If it is not, you have to do rebuilding. 
If the fit is correct, I would leave them as they are. Assuming the 
distribution of bond lengths and angles has a bell-shape, there will always be 
some residues at the extreme ends.

Best, Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Mittwoch, 17. Mai 2017 14:34
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Bond length and angle outlier fixing

HI all.

I am solving protein structure with 2.16A resolution. There are two chain in an 
asymmetric unit.
While submitted to PDB validation server, i could see few ligand bond -length 
and -angle outlier. Coot doesn't have any module that can help me with these as 
per my best understanding.
Kindly find the image of relevant details attached herewith.

Can somebody suggest me how to fix them?

Sincerely,
--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] AW: [ccp4bb] Poor density fit.

[Herman . Schreuder]

Dear Vipul,

the first thing I would check is why one chain has good and the other chain has 
poor side chain density. Are the B-factors of one chain much higher than of the 
other? Does one chain have more/better crystal contacts to stabilize its 
position in the crystal? Is the structure well-refined (e.g. R~20%; Rfree~25%)?

If this has all been checked, I would do as Bert suggested and leave the side 
chains intact and let the B-factors take care of the disorder. However, this is 
a very contentious issue, with probably 50% of the board members in favor of 
leaving the side chains intact and the other 50% in favor of truncating 
undefined side chains. Both approaches have their merits and I would do what 
you personally feel is the best, rather than setting off another mega-thread on 
this subject. You could try to google previous threads on this issue if you are 
interested! ;-)

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Donnerstag, 4. Mai 2017 17:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Poor density fit.

HI all.

I am solving protein structure with 2.16A resolution. There are two chain in an 
asymmetric unit. I see that in one of the chain, many residues' density for 
side chains is incomplete and therefore results in poor density fit.

I want to know your opinions for the approach I have taken. Figures relevant to 
each approach have been attached herewith.

Case1: There is no experimental density at all. Therefore, i have deleted side 
chains to Gly.
Case2: Though there is incomplete density for Leu, it is enough to suggest its 
rotamer. In this case, as may be seen, i have just set occupancy for atoms 
without density(CG, CD1, CD2) to zero.

Hopeful for the response.

--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] AW: [ccp4bb] Contouring 2Fo-Fc map, large blobs in Fo-Fc

[Herman . Schreuder]

Hi Roger,

First, sigma is a relative measure. If sigma is very low, e.g. since your map 
contains 80% solvent, 3 sigma may correspond to the same absolute value e.g. in 
electrons/Å3 as 1 sigma in a standard map, so I would not be worried about that.

However, an Rfree of 0.45 and a large off-origin Patterson peak indicates that 
you have to place another copy of your search molecule. This brings a few 
questions:

-Is the real space group P1, or is there higher symmetry? In the latter 
case you have to move to the higher symmetry space group.

-Did you ask phaser to search for 2 (or more) molecules? If the answer 
is no, that would be the first thing to do.

-Otherwise, I would translate the molecule already found according to 
the off-origin Patterson vector and look how this fits into the electron 
density maps.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Roger 
Shek
Gesendet: Mittwoch, 26. April 2017 03:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Contouring 2Fo-Fc map, large blobs in Fo-Fc

Hi everyone,

We have a structure that phaser found a solution for in P1, even though there 
is a large off-origin Patterson peak indicating pseudo NCS. However, the 
2mFo-Fc map needs to be contoured up to >3 sigma to be reasonable, going lower 
makes it look like what I normally see at <1 sigma. If you look at the Fo-Fc 
map, there are large areas of positive and negative density. The 2mFo-Fc map 
looks very convincing, you can see the tyrosines, phenyl rings, etc...Also 
phaser gave LLG of >400 and a TFZ of ~14. Rfree is ~0.45. Does anyone know 
what's going on?

Thanks,
Roger

--
Roger Shek

Stony Brook University
Graduate Student in Biochemistry and Structural Biology (PhD)
Stony Brook Integrative Structural Biology 
Organization
Cell: (808) 386-3879

[ccp4bb] AW: [ccp4bb] NCS difference

[Herman . Schreuder]

Dear Vipul,

At this resolution and with these Rfactors you are not supposed to „correct“ 
the NCS outliers. Look into the electron density maps if they are well defined 
and if the different conformations can be explained by e.g. crystal contacts.

However, if they are in a less well-defined region, you should superimpose the 
NCS symmetry molecule(s) and see which conformation would fit best and fit this 
conformation in the other molecules as well.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Montag, 24. April 2017 09:49
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] NCS difference

Hi all,

I am solving structure of one of the acyltransferse protein. We have collected 
data at 2.16A resolution. Currently the Rfree is 0.2508 and Rwork is 0.2042.

My query is regarding NCS difference. Under validation tool of coot while 
looking for NCS differene, i can find some residues with red bar. Can some one 
suggest me how may i minimize it if i am expected to do it?


--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] AW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Herman . Schreuder]

Dear Sutapa,

I fully agree with Grant, the first question is whether the naturally-produced 
protein is soluble and whether your protein is not a membrane protein, or a 
domain, cut out of a much larger protein? The other question is whether your 
protein is toxic for E.coli and only the bacteria producing it in inclusion 
bodies survive.

Another thing is to consider is to produce the protein in the same class of 
organism as the natural producer, e.g. prokaryotic proteins in a bacterial 
system, mammalian in proteins in mammalian cells etc.

My 2 cents,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Montag, 3. April 2017 09:06
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

There is also lack of information here.  Do you really expect this protein in 
soluble fraction? Is it a membrane associated or transmembrane protein?

Well, I don't have any experience in protein expression using codon 
optimization.
However, Considering the fact that in E.coli it is getting expressed in 
insoluble fraction, I would recommend to co-express this protein with groEL/ES 
system. I have plasmid expressing groEL/ES operon.

On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant 
> wrote:
Hi,

Why don't you try adding GST/MBP tags first? This is a easy quick test.

We have a nice fusion (MBP) vector for e.coli expression if you want.

Grant

From: Sutapa Chakrabarti 
>>
Reply-To: Sutapa Chakrabarti 
>>
Date: Monday 3 April 2017 08:49
To: 
"CCP4BB@JISCMAIL.AC.UK>"
 
>>
Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Dear All,

We're trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement.

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells.

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene?

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I'd like to know what the community thinks/has 
observed before I order an exorbitantly priced gene!

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094



--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] AW: [ccp4bb] Problem with MolRep

[Herman . Schreuder]

Dear Madhurima,

Small protein structures can be very difficult to solve by MR due to an 
unfavorable ratio between inter- and intra-molecular vectors in the pattersons. 
I am currently also struggling with some data sets myself.

However, there are things you could (and should) try:

1) If your search protein forms an oligomer, e.g. dimer, trimer etc., 
generate this oligomer and use this as a search model. Having a bigger search 
model will significantly increase your signal to noise ratio.

2) Try as many different MR programs as you can lay your hand on. Sometimes 
one succeeds, where others fail. I would at least try phaser.

3) Do not run the program(s) using the default parameters, but carefully 
read the manual and adjust the parameters based on the size and shape of your 
search protein. If your protein contains cysteines and/or methionines, you 
could look if there is any anomalous signal in your home source data. Getting 
higher resolution synchrotron data may not hurt as well.

Good luck!
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Madhurima Roy
Gesendet: Donnerstag, 9. Februar 2017 05:18
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Problem with MolRep

Hi all,
I have a small protein which is of 6 kDa including six histidine tag. The 
protein crystallized in the conditions given bellow :
a)  0.1M Sodium acetate trihydrate pH 4.6, 3M NaCl.
b)  0.1 M Bis-tris pH 5.5, 2M Ammonium sulphate.
The crystals are plate shaped in both conditions.We collected diffraction data 
at our home source using Rigaku RaxisIV and processed the data using XDS.

In spite of good homology, the protein structure cannot be solved by MolRep , 
the contrast is very low approx 1.65. The PDB Blast result is given below:

38.1(total score), 70%(query coverage) 1e-05 (E-value) and 50%(Identity) .
The screen shot of the statistics showing R factor and quality of fit in  
CORRECT.LP file is enclosed below.


Kindly help.

Thanks in advance.
Madhurima

[ccp4bb] AW: [ccp4bb] Composit omit map vs. ligand

[Herman . Schreuder]

Dear Petr,

Another possibility is that your very good substrate got turned over by the 
enzyme and that the 5 atoms with good electron density you see is all that is 
left. When you know the enzymatic reaction, this is easy to check. If this is 
the case, you should try a short soak (5-30 minutes) and immediately freeze the 
crystals. 

On the other hand, if your difference map shows reasonable density at the 1.5 
to 2-sigma level I would not worry too much. 

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Kay 
Diederichs
Gesendet: Mittwoch, 8. Februar 2017 11:43
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Composit omit map vs. ligand

Dear Petr,

if I understand correcty, the mFo-DFc density (1)  shows almost nothing, but 
the 2mFo-DFc  (2) as well as the composite omit map (3) show the ligand?

As you say, the apparent contradiction between (1) versus (2)&(3) is 
unexpected. One explanation could be that the Fc are simply too bad, i.e. the 
model not good enough to result in useful signal in the difference map.  OTOH, 
that you see the ligand in (2) may be simply model bias, so is not meaningful. 
(3) is hopeful since there is no model bias.

I would suggest to
- refine the occupancy, to find out why the density is so weak
- calculate a  (Fo,soak - Fo,native) (4) map with phases from a model unbiased 
by the ligand
- try a Polder map (5)

- If the occupancy is around 0.5 or higher, that would be a good sign. 
- but if you don't see density in (4) and (5), then your ligand is probably not 
there in any useful amount 

I consider (4) as the most sensible method to show presence of the ligand, and 
it should convince reviewers.

HTH,

Kay

 

On Wed, 8 Feb 2017 09:05:50 +0100, Petr Kolenko  
wrote:

>Dear colleagues,
>
>we have a dataset with potential enzyme:ligand complex at 2.2 AA 
>resolution. The ligand is very good substrate for the enzyme, we used 
>soaking. We do not see the ligand in the regular difference electron 
>density, only five out of twenty atoms. However, the ligand placed at 
>the active site (model used from structure of a mutant variant) is 
>refined well, giving no negative peaks in difference electron density 
>map and nice observed electron density. I have calculated composit omit 
>map with annealing in Phenix (input model did not contain the ligand) 
>and the electron density for the ligand is there.
>
>I have my own opinion, but we are desperate to obtain such data (more 
>than 40 crystals already tested). My question is, would this be proof 
>of presence of the ligand with reduced occupancy? Will this map 
>convince the reviewers? Is there any other way to validate presence of the 
>ligand?
>
>Best regards,
>Petr

[ccp4bb] AW: [ccp4bb] Unknown blob extended from catalytic serine residue

[Herman . Schreuder]

Dear Sharifah,

Was no protease inhibitor used during crystallization, or during the whole 
purification process? E.g. when a protease inhibitor cocktail was added during 
cell-lysis, a protease inhibitor may irreversibly have modified your serine.

If your protein is a protease, it may have reacted with a random peptide during 
purification and the modified protein may have selectively crystallized. In 
this case I would try to fit a few Ala residues as an acyl-intermediate, or 
even as a tetrahedral intermediate as the electron density seems to suggest, 
refine and see what side chains would fit best. You may also try mass-spec to 
get some information on the nature of your modification.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von sharifah 
nur hidayah syed mazlan
Gesendet: Samstag, 4. Februar 2017 16:03
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Unknown blob extended from catalytic serine residue

Dear All,

I am working on a structure with an unknown blob extended from the gamma O of 
the catalytic serine residue. The resolution of the dataset is 1.38 A. I have 
no idea whether the residue is modified or the blob belongs to other molecule.

The protein was expressed in Rosettagami (DE3), purified using Ni-Sepharose 
(affinity chromatography) and Q-Sepharose (Anion exchange chromatography). The 
crystallization formulation used contain 15% PEG 8000, 0.2 M Ammonium sulphate, 
0.1 M sodium cacodylate trihydrate pH 6.5; and the buffer composed of 50 mM 
Sodium chloride and Phosphate buffer pH 8. No protease inhibitor was used (eg: 
PMSF)

I have tried to fit in diethylene glycol as shown in one of the attached 
figures, but as observed, it is not really fit and the molecule is in incorrect 
conformation.

Kindly help me with this matter.



Thanks and regards,

Sharifah Nur Hidayah
Universiti Putra Malaysia,
Malaysia

[ccp4bb] AW: [ccp4bb] intermolecular dissulphides

[Herman . Schreuder]

Dear Eleanor,

I did not check the pdb file you mentioned, but I have had a case like that. 
The protein formed a complex of 8 large and 8 small subunits with internal 422 
symmetry. There was a disulfide link across the internal twofolds and in one of 
the crystal forms we got, this internal twofold came on top of a 
crystallographic twofold. At the time, I did not know about these sophisticated 
SSBOND records (if they existed at the time), so I assigned a very small van 
der Waals radius to the SGs to solve the repulsion problem.

So, if you have a dimer with an existing disulfide link across the twofold 
axis, this twofold axis may become a crystallographic twofold and there is no 
need for the disulfide bond to form afterwards.

Best regards,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Mittwoch, 1. Februar 2017 16:18
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] intermolecular dissulphides

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15
We seem to have one but it would have to form after crystalisation?
Eleanor

[ccp4bb] AW: [ccp4bb] Bad density for chains

[Herman . Schreuder]

Dear Pooja,

A few remarks:


-Matthews does not show 4 chains in the asymmetric unit, it suggests 4 
chains. However, in reality it can be more or less chains, although rare, 75% 
solvent (2 chains) is not unheard of.

-An initial Rfree of 38% is ok, 32% after refinement is a bit high

-Disordered loops do exist and you may have to live with them (not be 
able to build them).

-To correct for anisotropy, I suggest the staraniso server from global 
phasing.

-Unless you ran your molecular replacement in P1, Zanuda will confirm 
the space group you choose for MR. So run MR in P1 (if your symmetry is not too 
high) and run Zanuda again. (Have someone) look critical at the assigned space 
group and assess whether another choice might also be possible.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Pooja 
Kesari
Gesendet: Donnerstag, 26. Januar 2017 15:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Bad density for chains

We have a 2.6 A structure showing four chains in an asymmetric unit. Our 
protein is 360 residues around 40 kDa . Mattews shows four chain in an 
assymetric unit (solvent 49% mattews coeff 2.44). The template has about 60% 
homologous with our protein. The molecular replacement against this template 
gave an initial free R of 38.  We did chain tracing and found that we have good 
density (2Fo-Fc) for chain A and B but poor density for C and D.

1. The density for a particular stretch of 10 amino acids (disordered loop 
region) is absent in all the chains. We could not found density for this 
flexible loop region in any of the already known structures. Any suggestion on 
how can we build this region?

2. We did not find density for most of the loop regions in chain C and D which 
were well traced in chain A and B. How can we improving the density for these 
two chains based on chain A and B (Density modification)?

3. We analysed the data using phenix xtriage and found that our data shows 
severe anisotropy. Any suggestion of anisotropy correction?

Pointless and Ctruncate analyses didn't show twinning or NCS.  I have checked 
the space group using Zanuda. We are stuck at a free value of 32.

On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson 
> wrote:
This is a bit too vague to help much.
How did you solve the structure?
Eleanor

On 26 January 2017 at 03:50, Pooja Kesari 
> wrote:
Dear All,
Thank you all for reply.
We have checked the data for twinning.
Our protein is 360 residues around 40 kDa protein.
We have tried TLS refinement.
chain A and B don't superimpose well with chain C and D. (A and B chains also 
share slight difference )
Since we don't have proper density for some regions  chain C and D, we are not 
sure whether these chain have similar or different conformations.
We tried anisotropy correction and the model refined a bit.


On Wed, Jan 25, 2017 at 10:32 AM, Debanu 
> wrote:
Hi Pooja,

Are you positive you have the correct space group and there are no other issues 
like twinning, etc?

If sure, did you define NCS groups in refinement? TLS refinement? Try different 
refinement programs?

How big is the molecule? Was it solved by MR or experimental phasing?

You can try superimposing A/B on C/D and refinement with tight NCS then adjust 
NCS restraints during model adjustments based on local differences or also see 
if phenix autobuild helps.

Best,
Debanu
--
Debanu Das
Accelero Biostructures


On Jan 24, 2017, at 8:42 PM, Pooja Kesari 
> wrote:
Dear All,
I have a 2.6 A resolution structure having four chains in an asymmetric unit.
The chain A and B have density for almost all residues however we don't have 
proper residue density in chain C and D.What can be tried to build chain C and 
D ?


Many Thanks
Pooja



--
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA





--
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA

[ccp4bb] AW: [ccp4bb] Salt bridge-hydrogen bonds

[Herman . Schreuder]

Yes, Arg has 3 potential Hbond donor/acceptors and Glu 2. Also a single atom 
can have multiple interactions.
Look in coot at the structures and which atom has which interaction with which 
partner atom.

Best,
Herman

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mohamed 
Noor
Gesendet: Freitag, 20. Januar 2017 11:45
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Salt bridge-hydrogen bonds

Dear all

Is it possible for two residues to form a salt bridge between them, and at the 
same time**, each of them form a hydrogen bond with another residue? In other 
words:

Arg1 - Glu10 (salt bridge)

Arg1 - Tyr600 (H bond)

Glu10 - Thr590 (H bond)


** I understand proteins are not static structures, so I am referring to an 
exact time and space here and not something formed and broken throughout a 
sampled period of time.

[ccp4bb] AW: [ccp4bb] on space group

[Herman . Schreuder]

Dear Smith,

I think your question was clear, and the answer you got was clear as well.

However, I think the question you asked was not the right question. You want to 
use a particular phrase to describe your crystal packing and you want the 
CCP4BB to endorse this. When the answer was negative, you asked again the same 
question.

The real question, in my eyes, is “What is the best way to describe my P65 
crystal packing” since I guess you want to use this in your paper. Here I would 
use something like “in the crystal, the subunits are related by a 6-fold screw 
axis”. To be more precise, you could even mention a 65-screw axis. Other board 
members may even have better descriptions.

By the way, 61, 62, 63, 64 and 65 axes are all 6-fold screw axes, but of 
different types.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Lee
Gesendet: Donnerstag, 19. Januar 2017 06:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] on space group

Dear All,

Here may I make my question much clear? For the space group P 65 crystal, it 
seems we can call it "6-fold packing of subunits around a screw axis in the 
crystal". Then for the space group P 64 crystal, can it also be called "6-fold 
packing of subunits around a screw axis in the crystal"?

Smith

On Thursday, January 19, 2017 11:50 AM, Ethan Merritt 
> wrote:

On Thursday, 19 January 2017 02:33:14 AM you wrote:

>
> Dear All,
> In the literature, somebody call space group P65 crystal as  "six fold screw 
> axis crystal packing", then I would not make any mistake if I call P64 space 
> group crystal also as  "six fold screw axis crystal packing",am I right?
> I am looking forward to getting a reply from you.
> Smith


"six-fold screw axis" refers to the symmetry.

"crystal packing" refers to the molecule-to-molecule contacts regardless of 
symmetry.

So no, I don't think "six fold screw axis crystal packing" makes any sense.

--
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,  University of Washington, Seattle 98195-7742