Re: [ccp4bb] What could these crystals be?

[Antony Oliver]

Dear Careina,

Don't forget that the DNA you use is also a variable — you may need to make 
minor or indeed major tweaks to this is order to improve your crystals; e.g. 
subtract or add a base / base-pair.  Add a 1 base overhang etc..

Good luck!

Antony



- - - - - - - - - -
Dr Antony W. Oliver
Principal Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

antony.oli...@sussex.ac.uk (he/him)
https://tinyurl.com/aw-oliver

(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512






From: CCP4 bulletin board  on behalf of 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 24 November 2023 08:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] What could these crystals be?


I wanted to thank everyone for their suggestions and ideas regarding the 
eyeball shaped crystals that I got at the beginning of the month.
I can confirm that these crystals do indeed contain protein and DNA which is 
good news.
I have tried a number of different buffers and salts since then. I have also 
tried seeding but nothing I have tried has changed the morphology of the 
crystals. They remain thin, flat and eyeball/pumpkin seed shaped.
It is not difficult to make the crystals, they form quite easily under quite a 
few conditions. The difficulty is in the diffraction. By changing the buffer 
conditions we can now see some very weak diffraction (previously there was no 
diffraction at all).
Are there any suggestions as to how to improve diffraction of these crystals? I 
did try different cryoprotectants, parabar, glycerol, PEG but no difference. I 
think perhaps the problem is heterogeneity considering my sample contains both 
protein and DNA.
Any suggestions or thoughts are welcome.
Thank you
Careina

On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, careinaedgo...@yahoo.com 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:



The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG

Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP

The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution


On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, 
stephen.c...@rc-harwell.ac.uk  wrote:


Hi Careina,

Without knowing what's in your protein buffer or crystallisation condition it's 
hard to comment.

Best wishes,
Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

From: CCP4 bulletin board  on behalf of 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 08 November 2023 15:00
To: ccp4bb 
Subject: [ccp4bb] What could these crystals be?

Hi all
We have been trying with no success to crystalize a protein. Recently we got 
these strange shape "crystals". They are hard and flat but they do not diffract 
at all. Any ideas as to what could cause this?
Careina



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Re: [ccp4bb] What could these crystals be?

[Antony Oliver]

Looks like classic, poorly-diffracting “potato” crystals to me.
Will need additional optimisation to convince them into a nicer morphology.
No diffraction indicates that they are likely not to be salt at least.

Tony.


Antony W Oliver FHEA, PhD
Principal Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ





(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512

antony.oli...@sussex.ac.uk; (he/him)
https://tinyurl.com/aw-oliver
https://orcid.org/-0002-2912-8273


From: CCP4 bulletin board  on behalf of 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: 08 November 2023 15:00
To: ccp4bb 
Subject: [ccp4bb] What could these crystals be?

Hi all
We have been trying with no success to crystalize a protein. Recently we got 
these strange shape "crystals". They are hard and flat but they do not diffract 
at all. Any ideas as to what could cause this?
Careina



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Re: [ccp4bb] Baculovirus expression system

[Antony Oliver]

Dear Dhiraj,

It may be perceived as “slow”, but we (still) use the Bac-to-Bac system from 
ThermoFisher (previously Invitrogen).
This coupled with the pFastBac, MultiBac or biGBac vector systems generally 
works well;  some proteins/complexes may express better in High 5 cells over 
Sf9/Sf21.

With regards,

Antony.


Antony W Oliver FHEA, PhD
Faculty Senior Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ





(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512

antony.oli...@sussex.ac.uk; (he/him)
https://www.sussex.ac.uk/lifesci/oliverlab
https://tinyurl.com/aw-oliver
https://orcid.org/-0002-2912-8273
http://www.sussex.ac.uk/lifesci/internal/staff/support

From: CCP4 bulletin board  on behalf of "Srivastava, 
Dhiraj" 
Reply to: "Srivastava, Dhiraj" 
Date: Monday, 7 February 2022 at 16:24
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Baculovirus expression system

Hi All
 sorry for the question not related to crystallography. What is the 
baculovirus expression system that people use these days to get good yield in 
less time?

Thank you
Dhiraj





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[ccp4bb] Post Doctoral Position

[Antony Oliver]

A fixed-term (1 year in the first instance) Post-doctoral Research Fellow 
position is available in the laboratory of Dr. Antony Oliver and Prof. Laurence 
Pearl, working as part of an ongoing drug discovery programme that seeks to 
discover small-molecule inhibitors of selected therapeutic targets, as 
potential treatments for human disease.

We are seeking to appoint a Biochemist / Biophysicist / Structural Biologist to 
be responsible for the expression, purification, and crystallisation of 
selected targets — as well as performing a range of biochemical and biophysical 
assays. Applicants should have a PhD, with demonstratable experience in 
recombinant protein expression and purification. A background in structural 
biology / drug discovery is highly desirable.
For more details, and how to apply please see: 
https://www.sussex.ac.uk/about/jobs/research-fellow-ref-6401


Antony W Oliver FHEA, PhD
Faculty Senior Research Fellow
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ





(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512

antony.oli...@sussex.ac.uk<mailto:antony.oli...@sussex.ac.uk>
https://www.sussex.ac.uk/lifesci/oliverlab
https://tinyurl.com/aw-oliver
https://orcid.org/-0002-2912-8273
http://www.sussex.ac.uk/lifesci/internal/staff/support








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[ccp4bb] Apple Silicon / XQuartz / X11 / CCP4

[Antony Oliver]

Perhaps this is a little too soon, but does anyone know if the new M1 
system-on-a-chip will continue to run XQuartz/X11 + the CCP4 program suite + 
other crystallography / EM software? Will CCP4 continue to support the platform?

(a quick check of the GitHub page indicates that there is already an ARM64 
implementation of XQuartz)

I am not in a rush to purchase the newly announced computers, but am keen to 
understand if we are going to need to future-proof ourselves when the entire 
family moves over to the new hardware.

With thanks,

Antony.



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[ccp4bb] XCE - Refinement Fails

[Antony Oliver]

I am trying to convince XChemExplorer to refine my PANDDA models – i.e. those 
which have had ligands saved / placed into promising density. However, the 
large majority of refinement jobs (started by exporting all PANDDA models) 
simply fail.

Is there a log file or something I can look at to see where any obvious 
problems may have arisen?

With thanks,

Antony.

- - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow (Faculty)
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
United Kingdom

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl/aw-oliver

e: antony.oli...@sussex.ac.uk
t: +44 (0)1273 678349

Re: [ccp4bb] unusual monoclinic relation?

[Antony Oliver]

Dear Andrew,

If you're sub 2 Angstrom - give Archimboldo a shot. We recently solved the 
crystal structure of a unknown protein this way. You perhaps have the advantage 
of knowing what  be in there.

Antony.

--- Antony W Oliver ---
--- sent from my mobile account ---

On 20 Dec 2016, at 08:18, Andrew Lovering 
> wrote:

Thanks Tim,

Yes, I may give that a go next time (the wild-type was actually solved by 
S-SAD, very nice ordered bridge)

Orperhaps time to seed / derivitize / mutate and get different packing.
Still, not before xmas!

Andy

-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: 19 December 2016 19:58
To: Andrew Lovering
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] unusual monoclinic relation?

Dear Andrew,

did you remove all cysteins, and all methionines with the mutations? Your 
resolution is about 2A, if I understand correctly. This may be suitable for S- 
SAD. I would try to get access to a modern inhouse machine to get high quality 
data at high multiplicity. Some modern synchrotron beamlines offer more than 
1-circle goniometers, but good quality data is more easily collected with a 
multi-circle instrument.

Best,
Tim

On Monday, December 19, 2016 4:42:00 PM CET Andrew Lovering wrote:
Thanks again Herman,

The protein is a two domain protein (approx 40aa, 350aa split) -
searching with either is proving fruitless.

Original, wild-type cell = 49 x 75 x 80 P212121 This painful mutant =
39 x 157 x 75 beta=98.26 P21

(so one can say that there seems to be a relationship there, wt b =
mutant c, wt c = 2x mutant a?)

We have been bitten in the past by crystallizing contaminants, but
(touch
wood) those are always small crystals one / drop, in a few conditions.
This problem set has been replicated across at least 6 differing
crystals (grown in different conditions), where there are many
crystals / drop..along with the similar cell I'm confident that we
are seeing diffraction from what we expect

I will check the diffraction images more closely, but (does anyone
agree
here?) I find this sometimes obscured by modern fine-slice/Pilatus
methodology

Might be one for 2017! p.s. no anomalous signal in there - ironically
mutant we're using knocks bound metal out!

Thanks again
Andy
From: herman.schreu...@sanofi.com 
[mailto:herman.schreu...@sanofi.com]
Sent: 19 December 2016 16:26
To: Andrew Lovering; CCP4BB@JISCMAIL.AC.UK
Subject: AW: unusual monoclinic relation?

Dear Andy,

I don't think you will solve this pre-Xmas, but here are some more
suggestions: -is there any relationship with the unit cell of the
parent, unmutated protein? This might give some ideas of the packing
of the problem crystals. -are some promising solutions being rejected
due to clashes? In that case you might try to allow for more clashes.
-can the protein be split in some separate domains? In that case you
could try MR with the separate domains. -Are you sure the crystallized
protein is the protein you think you crystallized? (see contamination
database). -Check your diffraction images to make sure there are no pathologies 
present.

Best,
Herman


Von: Andrew Lovering [mailto:a.lover...@bham.ac.uk]
Gesendet: Montag, 19. Dezember 2016 12:30
An: Schreuder, Herman R/DE;
CCP4BB@JISCMAIL.AC.UK
 Betreff: RE:
unusual monoclinic relation?

Thanks Herman. In short:

-no twinning suggested from xtriage etc
-P2 doesn't give a solution either
-monoclinic cell would have 2 (60% solvent) or 3 (40% solvent) copies
-I originally thought the zanuda P1 route would be the way to go, but
phaser was still churning away after running overnight and so I killed
the job thinking it was thrashing

Andy

From: 
herman.schreu...@sanofi.com
[mailto:herman.schreu...@sanofi.com] Sent: 19 December 2016 10:43
To: Andrew Lovering;
CCP4BB@JISCMAIL.AC.UK
Subject: AW: unusual monoclinic relation?

Dear Andrew,

Just a few questions:
-Do the processing/refinement programs suggest twinning?
-Are you sure your space group is P21 and not P2? Did you try MR in P2?
-How many protein molecules do you expect in the asymmetric unit?

P2(1) is a very low symmetry space group. In this case I would not try
to be clever and just reprocess the data in P1 and run MR in P1. With
Zanuda you can afterwards try to figure out what the real space group could be.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
Andrew Lovering Gesendet: Montag, 19. Dezember 2016 09:43
An: 
CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] unusual monoclinic relation?

Dear All,

I have just collected data on a mutant of a protein that should 

Re: [ccp4bb] Modeling protein tertiary structure

[Antony Oliver]

Phyre2 absolutely deserves a mention. My 'threader' of choice.

--- Antony W Oliver ---
--- sent from my mobile account ---


On 10 December 2016 at 00:05, Myeongseon Lee (이명선) 
<0e01bd27cd0f-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Hi, all.

Could you anyone recommend me modeling programs for protein tertiary structure?

I have 3D strunture of a template protein and amino acid sequence of my 
protein. These two proteins are very close structurally and functionally.

I have done this kind of modeling with template 3D structure and amino acid 
sequence of an unknown structure long time ago for other project. But, 
unfortunately, I could not remember what program I have used at that time.

Have a great day.


Sent from my Samsung device

Re: [ccp4bb] What to interpret??

[Antony Oliver]

Oxidation of cysteine.

- - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
United Kingdom

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl/aw-oliver


e:  antony.oli...@sussex.ac.uk
t:  (office) +44 (0)1273 678349
t:  (lab) +44(0)1273 677512







On 01/02/2016, 08:53, "CCP4 bulletin board on behalf of dhaval patel" 
 wrote:

>Hi..Everyone
>
>There are blobs around few residues in the data (see attached images).
>Are they alternate form of residues??
>
>Kindly suggest

Re: [ccp4bb] ssDNA

[Antony Oliver]

1) Commercially made oligonucleotides
2) M13 rolling circle replication (how people used to make ssDNA for sanger 
sequencing).

———
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

e: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
t(office): +44 (0)1273 678349
t(lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver


[cid:EF31410E-E250-44AB-83E7-4BCEAF6FDAD0@gdsc.susx.ac.uk]

On 2 Jul 2015, at 16:40, Reza Khayat 
rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote:

Hi,

Sorry for the non-crystallography question, does anyone know how to produce 
milligram quantities of single stranded DNA? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 12318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

Re: [ccp4bb] Skin on drops

[Antony Oliver]

Have you tried crystallising in microbatch format, i.e. under oil? 

I've had success with this method for exactly the problem you describe. 

Regards. 

Antony

--- sent from my mobile account ---

 On 25 Feb 2015, at 08:39, Ulrike Demmer ulrike.dem...@biophys.mpg.de wrote:
 
 Dear crystallographers,
 
 I am trying to crystallize a soluble protein which tends to form aggregates. 
 The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the 
 crstallization process a thick skin is formed on top of the sitting-drops. As 
 well the crystals are buried in precipitate. Before I start harvesting I try 
 to remove the skin but still it is hardly possible to get any crystals out of 
 these drops.
 
 Any suggestions how to avoid the formation of skin on crystallization drops ?
 
 Cheers,
 
 Ulrike

Re: [ccp4bb] merging anisotropic datasets

[Antony Oliver]

Would the CCP4 program BLEND be a suitable initial option? And then the 
anisotropic server?

Tony.

--- sent from my mobile account ---

On 12 Nov 2014, at 21:29, Robert Keenan 
bkee...@uchicago.edumailto:bkee...@uchicago.edu wrote:



I have three datasets of varying quality collected from different regions of a 
single crystal. In each case, the data are anisotropic (from Aimless using 
CC1/20.5):

Dataset 1:  3.5, 3.5 5.5 A
Dataset 2:  4.2, 4.3, 4.8 A
Dataset 3:  3.7, 3.9, 4.4 A

I initially took a simple-minded approach and processed each dataset at the 
appropriate resolution limit (set 1: 3.5A; set 2: 4.2A; set 3: 3.7A) using 
XDS/XSCALE as implemented in xia2. The resulting merged dataset seems fine 
(albeit with ugly stats in the high-res bins because of the anisotropy), and it 
allowed me to solve the structure (Rfree/Rcryst ~31/26).

Now I am wondering if I can improve the maps further by first applying an 
ellipsoidal truncation (using the UCLA diffraction anisotropy server) and then 
scaling/merging the three datasets together. However, it seems that the UCLA 
anisotropy server only allows input of one dataset at a time, and it outputs 
merged amplitudes.

Is there some way to obtain the elliptically truncated but unmerged data for 
each of the three datasets?

More generally, are there preferred strategies for dealing with strongly 
anisotropic data?

Bob


Robert Keenan
Associate Professor
Dept. of Biochemistry  Molecular Biology
GCIS W238
University of Chicago
929 East 57th Street
Chicago, IL  60637
(o)  773.834.2292
(f)   773.834.5416
(e)  bkee...@uchicago.edumailto:bkee...@uchicago.edu
http://keenanlab.bsd.uchicago.edu

Re: [ccp4bb] Problem in molecular replacement

[Antony Oliver]

Try using DNA as a search model - this has worked very successfully in our 
hands before.

Tony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

On 24 Jul 2014, at 12:50, dusky dew 
duskyde...@gmail.commailto:duskyde...@gmail.com wrote:

DEAR ALL
I am trying to solve the structure of a protein DNA complex with molecular 
replacement. The resolution in about 2.5 angstrom.  The spacegroup is P21. The 
search model has about 50% identity and is a dimer of a dimer. The problem is 
the unit cell is huge and so the number of molecules in asu becomes 4 or 3. I 
am not finding any solution with phaser/molrep.
Please suggest.
Thanks
Yang zhang

Re: [ccp4bb] Unidentified density in coot

[Antony Oliver]

Dear Shanti

Looks like you’re looking down the symmetry axis - so this could simply be 
‘noise’, or a superposition of two bound ligands on top of each other… what’s 
your cryo-protectant?

With regards,

Tony.
- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

On 23 Jun 2014, at 13:17, Shanti Pal Gangwar 
gangwar...@gmail.commailto:gangwar...@gmail.com wrote:

Dear All

I have solved a structure of my protein at 3.0 A. The crystallization condition 
is consisting of PEG400, NaCl, MgCl2 and Sodium citrate. The protein was 
purified in HEPES buffer.
I can see an unidentified electron density blob in coot and I am not able to 
figure out what it could be?

I have attached the snapshot of that blob with this mail. I request everyone to 
please help me in identification of this blob.

Thanking you in advance.







Shanti Pal



Best regards
Shanti Pal Gangwar, Ph.D
School of Life Sciences
Jawaharlal Nehru University
New Delhi-110067
India
Email:gangwar...@gmail.commailto:email%3agangwar...@gmail.com


2.png1.png

[ccp4bb] Rotamer Selection in Low Resolution Data

[Antony Oliver]

Dear Crystallographic Community,

Apologies for the cross-posting, but I *do* routinely use programs from all 
three software packages.

I find myself refining a relatively low resolution structure (3.5 Angstrom) - 
with 8 molecules in the asymmetric unit.
Is there a *simple* automated way to place “optimal-fit to electron density 
side-chain rotamers into my model?
Preferably in an NCS-independant manner?

With thanks,
Antony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

Re: [ccp4bb] Rotamer Selection in Low Resolution Data

[Antony Oliver]

Hi Pavel,

Sorry… the current ‘triumvirate’ is, in no particular order:

CCP4, Phenix and Buster (Global Phasing).

Any suggestions would indeed be useful.

Many thanks,

Antony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

On 18 Jun 2014, at 17:13, Pavel Afonine 
pafon...@gmail.commailto:pafon...@gmail.com wrote:

Hi Antony,

Apologies for the cross-posting, but I *do* routinely use programs from all 
three software packages.

I find myself refining a relatively low resolution structure (3.5 Angstrom) - 
with 8 molecules in the asymmetric unit.
Is there a *simple* automated way to place “optimal-fit to electron density 
side-chain rotamers into my model?
Preferably in an NCS-independant manner?

naively assuming that one of the three software packages that you did not 
mention by name is Phenix:
yes, you can do it in a number of different ways. Let me know if interested and 
I will list all options.

Pavel

Re: [ccp4bb] Xia2 / XDS issues

[Antony Oliver]

Dear all,

Firstly - many thanks to Kay Diederichs and Graeme Winter.

The “memory” error was in fact due to certain diffraction images having very 
few (weak), if any spots - essentially they were ‘blank’.
By excluding these images, and with a combination of Xia2, plus XDS + Aimless, 
I was finally able to satisfactorily process the data.

FYI: The resultant mtz extends to ~ 3.6 Angstrom, with a CC(1/2) of 0.633.
Although the resolution isn’t the greatest, 8-fold NCS makes the resultant maps 
rather nice.

A great many thanks to others who offered useful tips and advice.

A slightly less tired Antony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

[ccp4bb] Xia2 / XDS issues

[Antony Oliver]

Dear CCP4-ers.

I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data.  

However, I am coming across the following issue, which I don’t seem to be able 
to solve...


 Integrating SWEEP1 
Status: error [XDS] cannot allocate memory”


I have checked XDS, and I have the correct (64-bit version installed: VERSION 
January 10, 2014  BUILT=20140307).
I also have set the parameter KMP_STACKSIZE to 8m, in my .bash_profile as 
indicated in the XDS setup instructions.
I am running OS X Mavericks (10.9.3) / XQuartz 2.7.6. 
I also have 16 GB of RAM installed.

Any help / suggestions would be very much appreciated.

Tony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - - 
email: antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

Re: [ccp4bb] Xia2 / XDS issues

[Antony Oliver]

Dear CCP4-ers.

Many apologies for the previous post (with attachments).

However, it is true -  somewhat crappy data, that I can’t seem to reprocess 
with XDS / xia2.

Tony.

HI Graeme,

Please find below, what I think it is that you need?  Files attached, plus 
relevant text clips,.

NB: everything seems to fail at the integration step.

NB2: This is quite crappy data….(!)

Tony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

[ccp4bb] Error using Coot supplied with CCP4

[Antony Oliver]

Dear CCP4 / Coot community,

I am trying to use LIDIA from Coot 0.7.2 - as supplied with CCP4-6.4.0 - to 
look at a protein / ligand complex.

However, when trying to launch LIDIA from the Extensions menu of Coot - I get 
the following error.

I guess this suggests some form of fortran compile error?

Any help would be gratefully received.

Antony.


(write-pdb-file 12 coot-ccp4/tmp-residue-for-prodrg.pdb)
arg_list ['XYZIN', 'coot-ccp4/tmp-residue-for-prodrg.pdb', 'MOLOUT', 
'coot-ccp4/.coot-to-lbg-mol', 'XYZOUT', 'coot-ccp4/.coot-to-lbg-pdb', 'LIBOUT', 
'coot-ccp4/.coot-to-lbg-lib']
forrtl: severe (180): SIGBUS, bus error occurred
Image  PCRoutineLineSource  
   
cprodrg000113FC  Unknown   Unknown  Unknown
cprodrg000111AC  Unknown   Unknown  Unknown
cprodrg00011164  Unknown   Unknown  Unknown
# BFONT COLOR=#FF!--SUMMARY_BEGIN--
# html !-- CCP4 HTML LOGFILE --
# hr
# !--SUMMARY_END--/FONT/B
# BFONT COLOR=#FF!--SUMMARY_BEGIN--


---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] changes in small sections of secondary structure

[Antony Oliver]

Dear Mahesh,

Are all the structures at similar resolution?  Definition of secondary 
structure is, and can be, affected by the level of geometric 
restraints/constraints used in the refinement process.

Tony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote:

 Hello experts 
 
 Thanks for your insights. 
 For one of the structures, it turned out to be a rendering issue by pymol 
 like matt pointed out. For the other, the residues are clearly in a less than 
 ideal position. Even if I see deviation from the RMSD plots, i cannot be sure 
 that the structure were refined ideally at those positions ( those are not my 
 structures, i just have the pdb files from my collaborator). 
 
 Thanks again, 
 
 Mahesh
 
 P.S from what all of you are saying it sounds like those changes are not 
 real, if I find that they could be Ill let everyone know. 
 
 
 
 
 

Re: [ccp4bb] Identity of a Bacterial lipid

[Antony Oliver]

Lipid or PEG from your crystallisation condition?

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Oct 3, 2013, at 1:42 PM, Andre Luis Berteli Ambrosio wrote:

 Dear colleagues,
 
 We have just determined the crystal structure (at 1.1 A max resolution) of a 
 recombinant protein that crystallized in complex with a leftover bacterial 
 lipid, the full identity of which we are currently struggling to identify. 
 Please see attached (3 views of the molecule).
 
 The map strongly suggests an 18-carbon long polyunsaturated fatty acid, with 
 5 conjugated unsaturations (at positions 5, 7, 9, 11 and 13, all cis), 
 covalently bound to some extra chemical group at is polar head. This extra 
 group seems to be comprised of 4 (5?) atoms, though I am afraid cannot tell 
 if it extends further into not-so-well-structured atoms.
 
 Myself and a student have spent the last two days searching on the web for 
 possible matches for this ligand without any success. For instance, we have 
 generated a SMILES formula for the aliphatic tail comprising the 
 unsaturations and browsed for similar compounds at PubChem with different 
 similarity cutoffs, but nothing seemed to resemble the complete molecule.
 
 We would appreciate if you could make any comments on the nature of this 
 ligand or perhaps suggest your favorite computational tools. We will perform 
 mass spec on it soon.
 
 Thank you beforehand.
 
 Andre LB Ambrosio, DSc
 Laboratório Nacional de Biociências - LNBio
 CNPEM, Brazil
  
 FA-density.png

Re: [ccp4bb] REFMAC5: linking a modified amino acid to adjacent residues

[Antony Oliver]

For REFMAC, I think you need to alter the cif file for the modified residue - 
such that the residue type is L-peptide rather than anything else.

Tony.

Sent from my iPhone

On 2 Aug 2013, at 17:08, Arnon Lavie la...@uic.edumailto:la...@uic.edu 
wrote:

Hi:

Despite Google and several attempts, I am still having no success in getting 
Refmac5 to link a modified amino acid to its adjacent residues.
Using sketcher, a cif file for the modified amino acid was generated.
This cif file was used an input to Refmac5.

The relevant part from the Refmac5 log file is shown below; note the not be 
used comment.


INFO: link is found (not be used) dist= 0.776 ideal_dist= 1.332

ch:BB res: 18 GLY at:C .-ch:Ba res: 19 THN at:N .

INFO: link is found (not be used) dist= 1.686 ideal_dist= 1.240

ch:BB res: 18 GLY at:O .-ch:Ba res: 19 THN at:N .


I also added the link comment in the pdb file as shown below:
LINKN THN B  19   C  GLY B  18
LINKC THN B  19   N  LEU B  20

Still, I fail to get a link between Gly18 to the modified residues THN19, and 
from it to the next residue Leu20.

What trivial mistake am I making?

Arnon



--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edumailto:la...@uic.edu
 http://www.uic.edu/labs/lavie/
***

Re: [ccp4bb] Heterogeneity during purification

[Antony Oliver]

Theresa,

Are there any cysteines in your protein?

Tony.

On 9 Jul 2013, at 05:01, Theresa Hsu theresah...@live.com wrote:

 Dear all
 
 I am working on a 30 kDa membrane protein which forms a functional dimer. The 
 protein is His-tagged at N-terminal. In small scale expression screening from 
 whole cells, there is only a single band on Western blot at 30 kDa. But, 
 after purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and 
 Western blot. On size exclusion with Superdex 200, a large proportion elute 
 near the void volume (8 ml).
 
 Detail purification
 
 For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
 mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 
 g. I then checked the supernatant on SDS-PAGE and scale it up for 
 purification.
 
 For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
 imidazole, 0.05 % DDM (two times CMC of DDM).
 
 Is there suggestion to get homogeneous protein?
 
 Thank you.
 
 Theresa

Re: [ccp4bb] Heterogeneity during purification

[Antony Oliver]

Theresa, 

I would suggest then, that you include reducing agent in your buffer (it 
doesn't appear to be to be there) - TCEP is good, as you can include in right 
from the start as it doesn't interfere with the IMAC columns.  My suspicion is 
that you are seeing multimerisation through inappropriate di-suphide formation.

Tony.


On 9 Jul 2013, at 08:18, Theresa Hsu theresah...@live.com
 wrote:

 Dear Tony
 
 Yes, there are four cysteines. 
 
 Theresa
 
 
 On Tue, 9 Jul 2013 06:26:19 +, Antony Oliver antony.oli...@sussex.ac.uk 
 wrote:
 
 Theresa,
 
 Are there any cysteines in your protein?
 
 Tony.
 
 On 9 Jul 2013, at 05:01, Theresa Hsu theresah...@live.com wrote:
 
 Dear all
 
 I am working on a 30 kDa membrane protein which forms a functional dimer. 
 The protein is His-tagged at N-terminal. In small scale expression 
 screening from whole cells, there is only a single band on Western blot at 
 30 kDa. But, after purification, additional bands appear at 60 and 120 kDa 
 on SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large 
 proportion elute near the void volume (8 ml).
 
 Detail purification
 
 For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
 mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 
 g. I then checked the supernatant on SDS-PAGE and scale it up for 
 purification.
 
 For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
 imidazole, 0.05 % DDM (two times CMC of DDM).
 
 Is there suggestion to get homogeneous protein?
 
 Thank you.
 
 Theresa
 
 

Re: [ccp4bb] Strand distorsion and residue disconnectivity in pymol

[Antony Oliver]

Er considering this forum, why not try CCP4MG ?

Tony.

On 30 May 2013, at 22:24, Petr Leiman 
petr.lei...@epfl.chmailto:petr.lei...@epfl.ch wrote:

On May 30, 2013, at 5:31 PM, Phoebe A. Rice 
pr...@uchicago.edumailto:pr...@uchicago.edu wrote:

In the olden days, when dinosaurs did roam, Ribbons had a nice fix-up feature 
that would gently move the alpha carbon end of the stick so that it met the 
ribbon.  Looked MUCH better than the current wavy ribbon one gets with side 
chain helper, and I haven't been able to find a trick like that in pymol.

So pymol developers, please consider yourselves wheedled ...



Well, there is another program, which is probably (no flames please!) more 
powerful, feature-rich than pymol and at the same time much easier to use 
called… drumroll… UCSF Chimera. One can display wavy and smooth secondary 
structure cartoons with it. The Chimera engine takes care of connecting side 
chains to the secondary structure cartoons in both representations.
The command to control the cartoon representation is in the Ribbon Style Editor 
menu:
https://www.cgl.ucsf.edu/chimera/docs/ContributedSoftware/ribbonstyle/ribbonstyle.html
The name of the command (can be run from the command line prompt in Chimera or 
chosen from the above menu) is ribspline:
https://www.cgl.ucsf.edu/chimera/docs/UsersGuide/midas/ribspline.html

Best wishes,

Petr

--
Petr Leiman
EPFL
BSP 415
CH-1015 Lausanne
Switzerland
Office: +41 21 69 30 441
Mobile: +41 79 538 7647
Fax: +41 21 69 30 422


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] 
on behalf of Jason Vertrees 
[jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com]
Sent: Thursday, May 30, 2013 10:17 AM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strand distorsion and residue disconnectivity in pymol

Hi Jacob,

Sure, but I believe Donghui wanted to only show sidechains, not any backbone 
atoms, as sticks. Doing this will leave fragments floating in space as the 
original email noted.

Cheers,

-- Jason


On Thu, May 30, 2013 at 10:13 AM, Jacob Keller 
j-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu wrote:

Can't you just show both cartoon (smoothed) and sticks (not smoothed) for the 
given area?

JPK


On Thu, May 30, 2013 at 11:06 AM, Jason Vertrees 
jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com wrote:
Hi Donghui,

Bernhard is correct: PyMOL flattens out secondary structure to produce more 
aesthetically appealing images. You can disable this for beta sheets and loops 
by typing:

# disable smoothing of sheets

set cartoon_flat_sheets, 0


# disable smoothing of loops

set cartoon_smooth_loops, 0


The cartoon_sidechain_helper setting automatically modulates these settings. If 
for some reason you need to disable cartoon_sidechain_helper you can imitate 
the look with:

hide
show cartoon
show sticks, not (n. C+CA+O+N) extend 1
set cartoon_smooth_loops, 0
set cartoon_flat_sheets, 0

Again as Bernhard noted, smoothing representations adjusts the representations' 
positions in space; therefore, you have the option of being positionally 
correct or aesthetically more pleasing.

Cheers,

-- Jason




On Wed, May 29, 2013 at 10:29 PM, wu donghui 
wdh0...@gmail.commailto:wdh0...@gmail.com wrote:
Dear all,

I found a problem when I use pymol to prepare structure interface. Strand is 
distorted when residue from the strand is connected to the strand by turning on 
side_chain_helper on. However when side_chain_helper is off, the strand turns 
to normal shape but the residue from it is disconnected to the strand. I 
attached the picture for your help. I know there must be some tricks for this. 
Welcome for any input. Thanks a lot.

Best,

Donghui



--
Jason Vertrees, PhD
Director of Core Modeling Products
Schrödinger, Inc.

(e) jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com
(o) +1 (603) 374-7120tel:%2B1%20%28603%29%20374-7120



--
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org

***



--
Jason Vertrees, PhD
Director of Core Modeling Products
Schrödinger, Inc.

(e) jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com
(o) +1 (603) 374-7120

Re: [ccp4bb] Negative FoFc around ligand

[Antony Oliver]

Dear Kavyashree, 

It is possible that your bound ligand (for which you have strong electron 
density) is actually a break-down of the parent compound? We have seen this a 
couple of times now. 

Also - are the poorly refining areas (those with negative density) part of a 
pendant ring connected by a conformationally unrestricted bond? These quite 
often have poor density. 

Hard to judge without seeing the actual density - but understand why! 

Regards, 
Antony. 


Sent from my iPhone

On 25 May 2013, at 10:40, Kavyashree Manjunath ka...@ssl.serc.iisc.in wrote:

 Dear Sir,
 
 Thank you all for your kind advice and clarifications.
 I will keep the occupancy 1.0 for both the ligands and
 refine without considering the negative density in this
 case.
 
 Thanking you
 Regards
 Kavya
 
 
 
 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1
 
 Dear Kavya,
 
 I don't see much sense in having different occupancies within the same
 molecule (unless one atoms sits on a special position, but then refmac
 will take care of it).
 
 If positive density comes up it's a good sign the ligand really is
 there. At 2.2A I would not be too surprised some atoms show less
 density than others (but don't look too much at the map with a sigma
 level  1: you are going to see what you want to see).
 
 It's difficult to judge without sitting next to you, so my advice is
 try to model it as good as your knowledge permits, and do take your
 chemical knowledge into account when doing so!
 
 Best,
 Tim
 
 On 05/25/2013 06:55 AM, Kavyashree Manjunath wrote:
 Dear users,
 
 I tried giving occupancy of 0.65 and 0.6 respectively for all atoms
 of the two ligands and refined. Now after refinement, the atoms of
 ligand does not have negative density but those which did not have
 negative density previously appear positive. So what do I need to
 do? under what circumstances does a ligand have different
 occupancies for different atoms or for a group of atoms. Any such
 references are very much welcome.
 
 Thank you Regards Kavya
 
 Sir,
 
 Yes it is around ligand. The average B-factor of one of the
 ligand is 36.78, of which one of the atom has occupancy B
 factor 0.58   39.37 0.56   38.77 0.87
 37.00 Three atoms are of same type. The other ligand's overall
 Bfactor is 17.64. occupancy B factor 1.0019.29
 0.6423.90 Two atoms are of same type.
 
 So in the present case should I put the occupancy of 0.56 for all
 atoms of ligand-1 and 0.64 for all atoms of ligand-2 and refine?
 
 I mean will it be wrong to put different occupancies for
 different atoms of same ligand?
 
 I could not see any alternate densities coming up for those
 atoms which did not have densities. But 2FoFc map would appear
 around these atoms at 0.7 sigma at the same position where the
 atoms are present.
 
 Thank you Regards Kavya
 
 Hi Kavya,
 
 If I understand you correctly (from title and text), you meant
 your negative FoFc was around your ligand, is that right? I
 wonder if this is a case in which the ligand has an occupancy
 below 1, but was modeled as 1, so the refinement program had to
 give it a high B factor to compensate that, which results in
 the electron density bleeding into nearby space where there
 should not be so much of it. If you want to test this
 hypothesis, one thing you can try is to set the occupancy to
 0.25, 0.5,0.75 and so on, and refine a few cycles to see what
 happens to the maps. Also, what's the B factor of the ligand,
 and what's the B of the nearby protein atoms? The difference
 between them can also give you some hint for guessing the
 ligand's occupancy. Some refinement programs(phenix.refine and
 shelx) can also let you refine the ligand's occupancy.
 
 As to the missing atoms, that could be caused by alternative
 conformations of the ligand - assuming you have already done a
 thorough refinement.
 
 Zhijie
 
 -- From:
 Kavyashree Manjunath ka...@ssl.serc.iisc.in Sent: Friday,
 May 24, 2013 12:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject:
 [ccp4bb] Negative FoFc around ligand
 
 Dear users,
 
 I am using refmac 5.7.0029 for refining a structure
 (resolution 2.2 Ang) bound to 2 ligands. After MR There is a
 very clear density of ligands but after refinement, I get
 negative fofc map near one of the ligand upto 5 sigma.
 However its 2fofc map covers the whole ligand. Also for the
 other ligand, I do not see any 2fofc density (at 3 sigma) for
 2 atoms, without these atoms the ligand is unrealistic. But
 the density comes up around these at around 0.7 sigma.
 Overall completeness is 99.9% Rmerge  7.5%
 
 What else I need to check in the data. Kindly provide some
 suggestions.
 
 Thanking you Regards Kavya
 
 
 
 
 
 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.
 
 
 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.
 
 
 
 -- 

Re: [ccp4bb] Negative FoFc around ligand

[Antony Oliver]

Kavya,

Presumably your enzyme is turning over the ATP? Driving it towards ADP?

In that case I suspect that you may have a mixture of ATP and ADP (and possibly 
contaminating AMP).
You could either model both ATP and ADP, and set relative occupancies of both 
(add up to 1).
Or, you could assign different occupancies to the phosphate groups alone.
I  personally think the former is probably the correct method? Comments CCP4 
community?

Many regards,
Antony.


On 25 May 2013, at 11:29, ka...@ssl.serc.iisc.in
 wrote:

 Dear Sir,
 
 That is true the ligand is ATP, the occupancy problem
 comes only for the phosphates. This ATP tends to get
 cleaved to ADP and AMP in other complexes I got. So in
 this case do you suggest keeping different occupancies
 for nucleoside and phosphate groups? I am not aware of
 any publication with this scenario so I am not sure whether
 it is right.
 
 
 Thank you
 Regards
 Kavya
 
 Dear Kavyashree,
 
 It is possible that your bound ligand (for which you have strong electron
 density) is actually a break-down of the parent compound? We have seen
 this a couple of times now.
 
 Also - are the poorly refining areas (those with negative density) part of
 a pendant ring connected by a conformationally unrestricted bond? These
 quite often have poor density.
 
 Hard to judge without seeing the actual density - but understand why!
 
 Regards,
 Antony.
 
 
 Sent from my iPhone
 
 On 25 May 2013, at 10:40, Kavyashree Manjunath ka...@ssl.serc.iisc.in
 wrote:
 
 Dear Sir,
 
 Thank you all for your kind advice and clarifications.
 I will keep the occupancy 1.0 for both the ligands and
 refine without considering the negative density in this
 case.
 
 Thanking you
 Regards
 Kavya
 
 
 
 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1
 
 Dear Kavya,
 
 I don't see much sense in having different occupancies within the same
 molecule (unless one atoms sits on a special position, but then refmac
 will take care of it).
 
 If positive density comes up it's a good sign the ligand really is
 there. At 2.2A I would not be too surprised some atoms show less
 density than others (but don't look too much at the map with a sigma
 level  1: you are going to see what you want to see).
 
 It's difficult to judge without sitting next to you, so my advice is
 try to model it as good as your knowledge permits, and do take your
 chemical knowledge into account when doing so!
 
 Best,
 Tim
 
 On 05/25/2013 06:55 AM, Kavyashree Manjunath wrote:
 Dear users,
 
 I tried giving occupancy of 0.65 and 0.6 respectively for all atoms
 of the two ligands and refined. Now after refinement, the atoms of
 ligand does not have negative density but those which did not have
 negative density previously appear positive. So what do I need to
 do? under what circumstances does a ligand have different
 occupancies for different atoms or for a group of atoms. Any such
 references are very much welcome.
 
 Thank you Regards Kavya
 
 Sir,
 
 Yes it is around ligand. The average B-factor of one of the
 ligand is 36.78, of which one of the atom has occupancy B
 factor 0.58   39.37 0.56   38.77 0.87
 37.00 Three atoms are of same type. The other ligand's overall
 Bfactor is 17.64. occupancy B factor 1.0019.29
 0.6423.90 Two atoms are of same type.
 
 So in the present case should I put the occupancy of 0.56 for all
 atoms of ligand-1 and 0.64 for all atoms of ligand-2 and refine?
 
 I mean will it be wrong to put different occupancies for
 different atoms of same ligand?
 
 I could not see any alternate densities coming up for those
 atoms which did not have densities. But 2FoFc map would appear
 around these atoms at 0.7 sigma at the same position where the
 atoms are present.
 
 Thank you Regards Kavya
 
 Hi Kavya,
 
 If I understand you correctly (from title and text), you meant
 your negative FoFc was around your ligand, is that right? I
 wonder if this is a case in which the ligand has an occupancy
 below 1, but was modeled as 1, so the refinement program had to
 give it a high B factor to compensate that, which results in
 the electron density bleeding into nearby space where there
 should not be so much of it. If you want to test this
 hypothesis, one thing you can try is to set the occupancy to
 0.25, 0.5,0.75 and so on, and refine a few cycles to see what
 happens to the maps. Also, what's the B factor of the ligand,
 and what's the B of the nearby protein atoms? The difference
 between them can also give you some hint for guessing the
 ligand's occupancy. Some refinement programs(phenix.refine and
 shelx) can also let you refine the ligand's occupancy.
 
 As to the missing atoms, that could be caused by alternative
 conformations of the ligand - assuming you have already done a
 thorough refinement.
 
 Zhijie
 
 -- From:
 Kavyashree Manjunath ka...@ssl.serc.iisc.in Sent: Friday,
 May 24, 2013 12:50 PM To: CCP4BB@JISCMAIL.AC.UK 

Re: [ccp4bb] Diffraction image viewer with display of resolution circles

[Antony Oliver]

iMosflm can certainly do this for you. You might have to screen grab to capture 
the resolution rings though. 

Tony. 

On 23 May 2013, at 09:27, Rafal Dolot rdo...@cbmm.lodz.pl wrote:

 Dear CCP4 users,
 
 I'm looking for the diffraction image viewer, which will be able to
 display of resolution circles and export it to new image. I tried use
 idiffdisp, but after choose of the Show/clear resolution circles, there
 is no action. Images were collected using Rayonics MX-225 detector - maybe
 detector format is a problem?
 
 Best regards,
 
 Rafal
 
 |--|
 |Rafal Dolot, Ph.D.|
 |  |
 |Polish Academy of Sciences|
 |Centre of Molecular and Macromolecular Studies|
 |Department of Bioorganic Chemistry|
 |Sienkiewicza 112  |
 |90-363 Lodz, Poland   |
 |Phone: +48(42)6803215 |
 |Cell:  +48 502897781  |
 |--|

Re: [ccp4bb] Why the name aimless

[Antony Oliver]

I thought it was just a logical pun progression from pointless to aimless.  

Perhaps the next program will be useless?

DISCLAIMER: This is intended as a JOKE...
I FULLY appreciate and use the wonderful programs generated by Phil.

Just in case I unintentionally start yet another CCP4bb flame-a-thon.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On May 2, 2013, at 11:23 AM, Martyn Winn wrote:

 I think Phil saw something nasty in the woodshed ...
 
 HTH
 Martyn
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Roberto Battistutta
 Sent: 02 May 2013 11:07
 To: ccp4bb
 Subject: [ccp4bb] Why the name aimless
 
 Hi everyone,
 just a curiosity, why the name aimless for the recent data reduction
 and analysis program in CCP4? You know, my students are curious ...
 Thank you,
 Roberto.
 
 
 Roberto Battistutta
 Associate Professor
 Department of Chemistry
 University of Padua
 via Marzolo 1, 35131 Padova - ITALY
 tel. +39.049.827.5262
 fax. +39.049.827.5829
 roberto.battistu...@unipd.it
 www.chimica.unipd.it/roberto.battistutta/
 VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129
 Padova - ITALY tel. +39.049.7923.236 fax +39.049.7923.250 www.vimm.it
 
 -- 
 Scanned by iCritical.
 

[ccp4bb] Curious electron density associated with Asp sidechain

[Antony Oliver]

Dear CCP4 colleagues.

I'm just finishing up a refinement, but am left with one little curio that I 
just can't seem to solve.

One aspartic acid residue is associated with some extra, unexplained electron 
density.

-- please see: http://i.imgur.com/vCYOqam.png

Where, the Fo-Fc map is contoured at 3.78 rsmd in Coot.

I have tried a number of different modelling scenarios, but as yet can't reach 
a wholly satisfactory conclusion; waters, alternate conformers, really don't 
seem to cut it.  I though about some radiation-induced phenomena, but this data 
set was collected on a home-source, so I guess this is unlikely.  

So, I would really appreciate some ideas and suggestions.  Hopefully it is 
blindingly obvious to someone.

Random Thought:  could it be PEGylation of the side-chain?  

Some other hopefully useful background information: 

* I'm sure it is/was an ASP, because the same protein (made from the same 
construct) has been used in previous crystallisations, and the resultant 
structures have clear, unambiguous electron density for the side chain.

* the crystallization condition is PEG 200, with some Na/K phosphate at pH 5.8, 
and NaCl.  The protein itself contains HEPES buffer. 

With many thanks,

Tony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] gelification of a pure protein

[Antony Oliver]

To ask a potentially daft question - but why do you need to reduce the salt?   
You say you are able to purify it, and that it behaves on gel filtration - but 
only starts misbehaving when you dialyse, and the NaCl is below 100 mM.

We've crystallised many proteins, particularly DNA-binding proteins with high 
pI, in high salt concentrations - even up to 1M.

Tony.

Sent from my iPhone

On 22 Apr 2013, at 23:36, Pascal Egea 
pas...@msg.ucsf.edumailto:pas...@msg.ucsf.edu wrote:

Dear All,

I am presently faced with a peculiar case in the lab. We are expressing a 
protein in E. coli and we are able to express it as a fusion protein without 
problems . Fusion cleavage goes well and the final product looks homogenous by 
size-exclusion chromatography with the expected molecular weight. There are no 
signs of aggregation. However when we lower the salt concentration by dialysis 
then the protein forms a gel. transparent , optically clear, with no fluffy 
material (in the cold room).

Gelification seems to occur when we lower the concentration below 100 mM NaCl. 
This protein has a fairly high pI (~9.0). Attempts to reverse the process by 
gentle heating or salt addition have been so far unsuccessful. It is not a 
thermophilic protein. We have not been able to obtain crystals so far.

Has anyone already observed this kind of behavior and/or have any suggestions?

Many thanks in advance .

--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515tel:%28310%29-983-3515
lab  (310)-983-3516tel:%28310%29-983-3516
email pe...@mednet.ucla.edumailto:pe...@mednet.ucla.edu

Re: [ccp4bb] CCP4 Update victim of own success

[Antony Oliver]

Eugene - that's great. I too run a small suite of Macs (12) and was trying to 
find a practical way of updating all those machines remotely. The command line 
version of CCP4um will be very useful. 

Many thanks. 

Tony. 

Sent from my iPhone

On 11 Apr 2013, at 18:19, eugene.krissi...@stfc.ac.uk 
eugene.krissi...@stfc.ac.uk wrote:

 Dear Dale,
 
 From next CCP4 release (due soon), ccp4um will be runnable from command line 
 in automatic, non-graphical mode, fully cronable. I hope that that will give 
 you what you want.  --check-silent is a special option for ccp4i, it only 
 checks for new updates but do not install them.
 
 Best regards,
 
 Eugene
 
 
 On 11 Apr 2013, at 18:10, Dale Tronrud wrote:
 
 FYI
 
  I have a small herd of computers here and find it cumbersome to ssh
 to each and fire up ccp4i just to update the systems.  ccp4i takes a
 while to draw all those boxes (particularly over ssh) and leaves files
 behind in my disk areas on computers that I'm not likely to, personally,
 run crystallographic computations.  I much prefer to simply run ccp4um
 from the command line.
 
  In fact, I would rather put it in cron and forget about it -- and
 I expect that is what --check-silent is for.  The usage statement,
 however, doesn't explicitly say that this installs the new updates it
 finds.  I'll have to experiment a bit.
 
 Dale Tronrud
 
 On 04/11/2013 05:17 AM, eugene.krissi...@stfc.ac.uk wrote:
 Sorry that this was unclear. We assume that updater is used primarily from 
 ccp4i, where nothing changed (and why it should be used from command line 
 at all ?:)). The name was changed because it is reserved in Windows, which 
 caused lots of troubles. Now it will stay as is.
 
 Eugene
 
 On 11 Apr 2013, at 05:16, James Stroud wrote:
 
 
 On Apr 10, 2013, at 9:30 PM, 
 eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk 
 eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk wrote:
 
 No, it got renamed to ccp4um :) That should have been written in update 
 descriptions, was it not?
 
 
 There was only one mention of ccp4um that I could find in all update 
 descriptions that I found (6.3.0-020). I only figured out what information 
 was trying to be communicated because of your message (see attachment).
 
 James
 
 
 um-what.png
 
 
 
 On 11 Apr 2013, at 03:54, James Stroud wrote:
 
 Hello All,
 
 I downloaded a crispy new version of CCP4 and ran update until the update 
 update script disappeared. Is the reason that CCP4 has reached its final 
 update?
 
 James
 
 
 -- 
 Scanned by iCritical.
 

[ccp4bb] Post-Doctoral Research Fellow - University of Sussex

[Antony Oliver]

A Cancer Research UK-funded Postdoctoral Research Fellow position is 
immediately available in the laboratory of Professor Laurence Pearl FRS and Dr 
Antony Oliver, to study the structural basis for the assembly and specificity 
of multi-protein complexes involved in the recognition and repair of DNA 
damage, and DNA damage signalling. 

The internationally renowned MRC Genome Damage and Stability Centre and the 
School of Life Sciences are very well equipped for all aspects of modern 
structural biology, with state-of-the-art laboratories for molecular biology, 
recombinant expression in bacterial and eukaryotic hosts, biochemistry, 
biophysics and X-ray crystallography. 

Excellent synchrotron access (~ 2 days/month) is also available through rolling 
beam-allocation programmes at both the Diamond Light Source and ESRF.  

Applicants must have a PhD, and extensive experience in recombinant expression 
and protein purification. Previous experience of crystallization and X-ray 
crystallography would be a distinct advantage. 

The post-holder will be responsible for expression, purification, 
crystallization and structure determination of protein-protein and protein-DNA 
complexes, plus downstream biochemical and biophysical characterisation. 

The Genome Damage and Stability Centre is an internationally renowned Institute 
carrying out research on the response of cells to DNA damage, genome 
instability and its relationship to human disease. We provide a stimulating and 
supportive environment and our expertise covers a range of experimental 
systems. Further information about our research can be obtained by visiting our 
website at:  http://www.sussex.ac.uk/gdsc

Fixed term for three years, full time.
Salary range: starting at £30,424 and rising to £36,298 per annum, commensurate 
with level of experience.
Closing date for applications: 17 May 2013
Expected start date: 1st June 2013 or as soon as possible thereafter.

===

Please note that previous applicants for Job Reference 768, advertised in 
August 2012, should not reapply.

Full information and instructions for APPLICANTS can also be found at the 
following URL:   

http://www.sussex.ac.uk/aboutus/jobs/090



WORD format Application Form:
http://www.sussex.ac.uk/humanresources/documents/acadnw.doc

PDF format Application Form:
http://www.sussex.ac.uk/humanresources/documents/acadnw.pdf

Application forms should be completed and sent via e-mail to: 
lifescirecruitm...@sussex.ac.uk

When emailing your application please use the following format in the 'subject' 
line: (post reference number / post title / your name). If you are applying for 
more than one post advertised by the University, please send a separate email 
and application form for each post. Please attach your completed application 
form and any other documents directly to the email rather than using a 
web-based upload / weblink service (e.g. SkyDrive) otherwise we may not receive 
your application due to incompatibility with our email software.

Alternatively, completed applications forms may be posted to: 
Human Resources Division, Sussex House, University of Sussex, Falmer, Brighton, 
BN1 9RH.

Applications should be received by midnight on the closing date.

INFORMAL enquiries ONLY may be made to Professor Pearl: 
laurence.pe...@sussex.ac.uk, or Dr Oliver: antony.oli...@sussex.ac.uk  

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom,

Q1: Are you really supposed to posting this information here?
Q2: Is there really NOT anyone you can ask in your own department to help you 
with this?

Some initial hints/ideas/suggestions though - although I actually assume this 
is meant to be a learning exercise for you …?

1) Have you got the correct spacegroup?
2) Have you got enough molecules in the asymmetric unit?
3) Do your placed molecules pack together to form a sensible crystal lattice?

Tony.

On 27 Mar 2013, at 16:22, Tom Van den Bergh 
tom.vandenbe...@student.kuleuven.bemailto:tom.vandenbe...@student.kuleuven.be
 wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of mRFP 
(monomer red fluorescent protein, sequence in attachment). I have done 
molecular replacement with phaser with model 2VAD of protein database. Then i 
have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
flags and log file attached) When i refine with phenix.refine my structure i 
get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, 
.mtz and logfile attached) When i look at the structure in coot i find many 
unmodelled blobs and many outliers in density analysis and rotamer analysis. 
The problem is that there are so many problems with my structure, that i dont 
know where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too many 
experience with it.

Greetings,

Tom

overall_best.pdbexptl_fobs_phases_freeR_flags.mtzoverall_best_placed.pdbredfluorescentprotein_refine_10.pdbredfluorescentprotein_refine_10.mtzmRFPsequentiezondertag.fastaredfluorescentprotein_refine_10.logAutoBuild_run_6_1.log

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

At the risk of being somewhat cheeky - perhaps I could claim second author?
I too have successfully solved the structure - and I totally concur with Phil.
Placing a second molecule in the asymmetric unit, essentially resolves the 
perceived R-factor problem.

A good thorough manual inspection and rebuilding is *ALWAYS* good practice for 
newcomers to the field.

Tony.


On 27 Mar 2013, at 17:14, Petr Leiman petr.lei...@epfl.ch
 wrote:

 Since this is now public domain knowledge and if this gets ever published, 
 Phil has my vote to be the first author!
 
 Petr
 
 
 On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
 
 That's quite brave - shipping your entire structure to people that could be 
 actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
 
 Practical points:
 
 * not everyone loves 12Mb of attachments in one email in their inbox, so if 
 you do this again please put the files on a webserver and point us there
 
 Structural points:
 
 * the map looks pretty good, but I think the sequence is misassigned in some 
 regions (e.g. A118-A122 etc).  Automation is a good tool but a poor master, 
 and extreme caution is required before taking the results too literally.  
 Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently 
 had a sequence misassignment at just that resolution. That map was trivial 
 to interpret with the correct sequence however - one of the joys of working 
 with Arp/wArp at 1.4 Angstrom.
 
 * the large number of positive difference density blobs and water molecules 
 clustered in what otherwise would be the solvent void strongly suggest that 
 there's a second molecule present.
 
 
 If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
 exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
 it finds them quite successfully.  (for the record an LLG of 15111 using 
 nominal sequence identity of 90%).  I will send this to you off-list.  
 Please note that Phaser is using a different origin for this molecular 
 replacement solution so the coordinates and your previous map do not overlap.
 
 This rather nicely explains why your structure had an R-factor in the 40's 
 despite being a half-way decent model.  The new MR solution has an R-free in 
 the 30's in the phenix.refine job I'm running right now.
 
 
 Going forward I suggest you utilize the Arp/wArp program to autobuild your 
 structure for you, starting from the molecular replacement solution (or, 
 perhaps with it stripped to ALA).  While you could use Autobuild, this is 
 the CCP4 list and so you should use CCP4 programs.
 
 Phil Jeffrey
 Princeton
 
 
 On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of
 mRFP (monomer red fluorescent protein, sequence in attachment). I have
 done molecular replacement with phaser with model 2VAD of protein
 database. Then i have done some model building phenix.autobuild. (2
 pdb's (overall...), freeR flags and log file attached) When i refine
 with phenix.refine my structure i get a R-value of 0,42 which is still
 way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
 When i look at the structure in coot i find many unmodelled blobs and
 many outliers in density analysis and rotamer analysis. The problem is
 that there are so many problems with my structure, that i dont know
 where to begin. Could you try some refinement for me, because this is
 first structure that i need to solve as a student and i dont have too
 many experience with it.
 
 Greetings,
 
 Tom
 

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom, 

I think that we've all actually been rather gently teasing you - admittedly a 
difficult concept to put across via the medium of e-mail.
In fact, I think we've all offered sensible constructive suggestions, and 
indeed pointed out what you should try next.

Apologies for any inadvertent offence - none intended. 

Tony.


 Dear so-far-posters,
 
 I do not know Tom Van den Bergh, nor do I know his background, nor the
 history of the data, nor the reasons why he may have sent it to this
 list (although I think he did it to ask for help), but I find these
 answers irritatingly disrespectful and nasty.

harsh

 No regards to the ones addressed,
 Tim

/harsh

Re: [ccp4bb] delete subject

[Antony Oliver]

Dear Tom,

I'm sure the files can be easily removed from the server, if that is what you 
wish / want to happen. A quick email to the administrators at 
c...@ccp4.ac.ukmailto:c...@ccp4.ac.uk should do the trick.

Reading around all the all leg-pulling / other comments aside - from your 
post you've got actually got a really good number of useful suggestions and 
comments that should help you along the path to solve and refine your structure 
yourself.

Best of luck with your data.  With regards,

Tony.



Is it possible to delete my post: refinement protein structure from ccp4 bb, i 
get too many bad reactions. I think its bettter to just delete the whole topic.

Greetings,

Re: [ccp4bb] Scaling with SCALA high and low resolution data sets

[Antony Oliver]

Or, seeing that this is the CCP4BB smile, you could use the Xia2 pipeline 
from CCP4i - which will also use XDS if installed on your system.

 My recommendation would be to process with XDS. By using the autoProc 
 procedure from Global Phasing this is very easy, even for people who are 
 normally not able to run a program without a GUI. You will then have to run 
 XSCALE manually, which is again trivial once XDS had run correctly.
 
 Good luck!
 Herman


Tony.


On 21 Mar 2013, at 07:41, herman.schreu...@sanofi.com
 wrote:

 Dear Kyriacos,
 
 
 
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
 Kyriacos Petratos
 Sent: Wednesday, March 20, 2013 6:25 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Scaling with SCALA high and low resolution data sets
 
 Dear All,
 
 we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a 
 single crystal.
 Integration with iMosflm seems to be fine like the scaling within each of the 
 data sets.
 When we try to merge and scale both of them with 'Scala' we get extremely 
 high scale factors for the lower resolution images varying between 
 approximately 30 and 200!
 Do we need to pay attention to some particular options for running the 
 program(s)?
 Thank you,
 
 Kyriacos
 e-mail: petra...@imbb.forth.gr

Re: [ccp4bb] laboratory phage infection [SEC=UNCLASSIFIED]

[Antony Oliver]

Dear Aidong, 

Some thoughts... 

Are you sure the problem is actually phage-related? I ask because you are still 
having problems using phage-resistant stains...

Acid-washing your glassware, and a long-dry autoclave sterilisation (as 
previously suggested) should really do the trick. A good *manual* wash-out of 
your incubators would also certainly not hurt. 

Clean / replace any filters in the incubators *and* Air-con if you have it!  
It's amazing what can creep in there...

Residual detergent in glassware can cause bacterial lysis. 

Also confirm that your autoclaves are actually reaching and maintaining their 
temperature during media / glassware sterilisation. 

Apologies if you've already tried all this!

With regards, Antony. 



Sent from my iPhone

 Dear Aidong,
 
 we never had that problem but I heard that only long dry(!) heat treatment of 
 glassware destroys the phages.
 No need to say that you have to do it with ALL your glassware.
 
 On 3/2/2013 12:15 PM, aidong wrote:
 Many thanks for your rapid inputs.
 
 We used to make glyerol stocks but now we do not since the protein 
 expression is not stable.  Our lab is about 10 students, who have to make 
 their own proteins so our three large-scale shakers are very crowded every 
 day.  We did try a formaldehyde gas treatment one time during our new year 
 leave, however, the situation did not seem to improve.  Since that gas is 
 much harder to handle, we only stick to ozone treatment.  Intriguingly, our 
 neighbor lab, also a crystallography lab, which constantly uses bacterial 
 cultures although not as much as we do, is just fine.
 
 Chloroform/phenol treatment might be a good one because some of our 
 constructs are just very difficult to get any transformed colonies while 
 some others are so easy.  We have used gel extraction method to purify our 
 plasmids because we thought some phage DNA is contaminated. This method 
 slightly improved our situations but not complete.  But I am thinking the 
 entire phages are simply there in plasmid preps purified through miniprep 
 columns.
 
 Cheers
 
 Aidong
 
 
 On Mar 2, 2013, at 7:30 PM, DUFF, Anthony wrote:
 
 Hi Aidong,
 
 some ideas...
 
 
 Do you use glycerol stocks of transformed expression cells (phage risk), or 
 do you do fresh transformations each time?
 
 Do you have many people working together, or do you have periods of 
 bacterial growth separated by periods of inactivity?
 
 Do you have neighboring labs who may be working with, or infected with phage?
 
 Do you have any old stocks, liquid media, antibiotics, etc, that could be 
 contaminated?
 
 sincerely,
 
 Anthony
 
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of aidong 
 [a...@xmu.edu.cn]
 Sent: Saturday, 2 March 2013 7:50 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] laboratory phage infection
 
 Hi All,
 
 Really sorry for my non-CCP4 related question.  Our lab mainly uses
 bacterial cultures to produce targets proteins for crystallizations.
 However, we have been struggling with phage infections to our
 bacterial cultures for a quite long time. To control its devastating
 effects, we have regularly been using large-dose ozone treatments on
 the whole lab space.  We have also tried anti-phage BL21/DE3 strains
 from Sigma USA but found it was still not avoidable. The lab has been
 maintained well hygienic and its outdoor environment is clean and
 neat. We keep good ventilations, including windows and central AC
 systems.  However, phages are still eating up our cultures with very
 low percentage of survivals. Therefore, this has been our big
 headache. We wonder whether you have the same experience and how to
 keep a lab free of those bugs. Your suggestions are deeply
 appreciated.  Thanks.
 
 Sincerely
 
 Aidong
 
 Aidong Han, Ph.D
 
 Department of Biomedical Sciences
 School of Life Sciences
 Xiamen University
 3 South Xiangan Road
 Xiangan, Xiamen 361102
 China
 Phone: 0592-218-8172 (O)
  0592-218-8173 (L)
 Web: http://life.xmu.edu.cn/adhanlab/
 
 Aidong Han, Ph.D
 
 Department of Biomedical Sciences
 School of Life Sciences
 Xiamen University
 3 South Xiangan Road
 Xiangan, Xiamen 361102
 China
 Phone: 0592-218-8172 (O)
  0592-218-8173 (L)
 Web: http://life.xmu.edu.cn/adhanlab/
 

Re: [ccp4bb] Unknown density

[Antony Oliver]

Dear Pavel,

Is this density located on a symmetry axis?  
There is quite often a lot of noise around these regions - and it may not, in 
fact, be possible to model this satisfactorily.

Tony.


---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Feb 25, 2013, at 10:03 AM, Natashin Pavel wrote:

 Hello everyone,
 
 
 I’m working on a structure with a 1.72Å resolution data and almost finished 
 it. But there is a piece of unknown density between two protein molecules. 
 The following pictures show this density from two sides:
 
 http://www.4sync.com/photo/WHewU7Ek/Density_1.html
 
 
 http://www.4sync.com/photo/RM3IEesw/Density_-_2.html
 
 This density is located between two protein molecules and is not connected to 
 either of them.
 
 I have tried to fit several small organic molecules from crystallization 
 conditions and protein sample buffer, also tried ligand database search using 
 “Phenix ligand identification” software, but no satisfactory results. 
 Crystallization condition: DL-Malic acid
 
 Sample Buffer: Bis-Tris, EDTA
 
 Reagents from protein purification step (also checked): Tris, Urea, 
 Triton-X100, DTT. 
 
 
 
 I will very much appreciate to hear any suggestions and ideas on what to do.
 
 Best regerds, 
 
 Pavel V. Natashin
 
  
 PhD student
 Photobiology Laboratory 
 Institute of Biophysics
 Russian Academy of Sciences Siberian Branch 
 Krasnoyarsk 660036, Russia 
 
 National Laboratory of Biomacromolecules
 Institute of Biophysics Chinese Academy of Sciences
 Beijing 100101, China

Re: [ccp4bb] off topic: DSSP

[Antony Oliver]

Sorry to cross-pollute…

If you don't mind using the ksDSSP implementation, it is already installed with 
the phenix suite if you have it.

phenix.ksdssp from the command line.

With regards,

Tony. 

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Jan 28, 2013, at 3:25 PM, Jon Agirre wrote:

 Dear Rashmi, 
 
 you may want to try this web server instead of compiling  installing it: 
 http://www.cmbi.ru.nl/hsspsoap/
 
 Otherwise, you'd better follow Tim's advice and give us more clues!
 
 Jon
 
 2013/1/28 Tim Gruene t...@shelx.uni-ac.gwdg.de
 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1
 
 Dear --
 rashmi,
 
 if you would kindly let us know what exactly you are thanking for, you
 may actually have a reason for doing so - a good start is a
 cut-and-paste copy of the error message(s) from the terminal where you
 typed 'make' and 'make install'.
 
 If you get this error only from 'make install' but not from 'make',
 chances are that the compilation worked fine but the Makefile attempts
 to install file to a location you do not have write access to.
 
 Cheers,
 Tim
 
 On 01/26/2013 10:11 AM, Rashmi Panigrahi wrote:
  Hi All Apologies for the off topic. Could some one help me in
  installing DSSP in Mac OSX 10.5.8 I tried downloading
  dssp-2.0.3.tbz
  ftp://ftp.cmbi.ru.nl/pub/software/dssp/dssp-2.0.3.tbz
  dssp-2.0.4-linux-amd64ftp://ftp.cmbi.ru.nl/pub/software/dssp/dssp-2.0.4-linux-amd64
 
 
 dssp-2.1.0.tgz ftp://ftp.cmbi.ru.nl/pub/software/dssp/dssp-2.1.0.tgz
  from http://swift.cmbi.ru.nl/gv/dssp/ I tried doing make followed
  by make install. I got error on compiling. Thanks in advance
 
 - --
 - --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen
 
 GPG Key ID = A46BEE1A
 
 -BEGIN PGP SIGNATURE-
 Version: GnuPG v1.4.12 (GNU/Linux)
 Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
 
 iD8DBQFRBpI8UxlJ7aRr7hoRAu1bAJ9q3TWZbBMAlrOWchZnrPo4Iy42YQCg1K+c
 uo0hlOQ0oTPyTDHmXjM57Bw=
 =dBMv
 -END PGP SIGNATURE-
 
 
 
 -- 
 Jon Agirre, PhD
 Unit of Biophysics (CSIC-UPV/EHU)
 http://www.ehu.es/jon.agirre
 http://sourceforge.net/projects/projectrecon/
 +34656756888

Re: [ccp4bb] Mac mini advice

[Antony Oliver]

Dmitry... everyone is of course entitled to their opinion - but as one of the 
brain-washed masses I feel I need to at least reply (!). Sorry Cara...

 I am not happy with the direction OS X is going. Too much emphasis on eye 
 candy and not enough on underlying technology.

Fair enough, but it does seem to be the way that most of the UIs/OSes are 
going. Erm, Windows 8, Unity...

Also Apple are *constantly* innovating under the hood - albeit mainly hardware 
- Lightning connectors, Fusion hard-drives etc... so they are one of the few 
computer manufacturers actually innovating at all(!)

 ZFS (long ago), Xgrid and X11 have been ditched, which I find disturbing. I 
 don't see Apple investing in computers given current revenue from that 
 sector.

I sort of agree with you here - but on the other hand at least it is still a 
Unix variant underneath and we still have Xquartz.  X11/X window is itself 
becoming quite an old beast and I'm pretty sure there's ever going to be an 
X12. Hence the probable rise and prevalence of Qt - so you can run any OS you 
like...

 Linux in a virtual machine of your choice might be a better bang for the 
 buck. Or, Windows in a virtual machine on a Linux box for that matter.

Or, ( controversially ) a Mac running Linux and Windows in virtual boxes !?

Tony.

Sent from my iPhone

On 23 Jan 2013, at 03:03, Dmitry Rodionov 
d.rodio...@gmail.commailto:d.rodio...@gmail.com wrote:

AFAIK there is no problem mixing and matching different timing RAM: system will 
run at the speed of the slowest module.
I don't think anybody will notice the difference with CAS latency Coot'ing and 
Refmac'ing.

I don't think there is much sense in having more than 4 GB of RAM per physical 
core on a Mac.
Majority of the Mac flock does not really care for where the RAM modules come 
from.
As for Mac Pro's- they use ECC RAM with proprietary heat sensors, so that's a 
completely different story. You can still use generic ECC RAM in a MAC PRO at 
the cost of the fan being stuck in hurricane mode.

The bottleneck of pretty much any modern system is the HDD. Apple-branded HDDs 
were known to have somewhat modified firmware, causing problems at times 
(mostly with AppleRAID, if not using an Apple-branded HDD)
An end user most definitely will notice an SSD VS HDD, which brings up TRIM 
support on OS X, which is limited to controllers sold by Apple.

Upgradeability-wise Apple is not the way to go in any case.

DISCLAIMER:  The rest may be much more inflammatory.

Personally, I am not convinced OS X and Apple is the way to go log term (having 
been surrounded by MACs for the past 4-5 years)
I am not happy with the direction OS X is going. Too much emphasis on eye candy 
and not enough on underlying technology.
ZFS (long ago), Xgrid and X11 have been ditched, which I find disturbing. I 
don't see Apple investing in computers given current revenue from that sector.

Linux in a virtual machine of your choice might be a better bang for the buck. 
Or, Windows in a virtual machine on a Linux box for that matter.

Don't kick me,
DIR



On 2013-01-22, at 7:22 PM, Bryan Lepore 
bryanlep...@gmail.commailto:bryanlep...@gmail.com wrote:

On Tue, Jan 22, 2013 at 1:40 PM, Phil Jeffrey 
pjeff...@princeton.edumailto:pjeff...@princeton.edu wrote:
I don't think that anybody has shown a significant performance difference on 
Apple memory vs a reasonable 3rd party supplier.  Apple may potentially have 
better quality controls but places like Crucial essentially have lifetime 
warranties on their memory.  I use Crucial at home and at work. [...]

sure, I agree with all this

the only other point I really wanted to make is to be cautious when configuring 
a computer on the Apple website, where they might say for memory DDR3 ECC 
SDRAM (checked this for a Mac Pro just now) but that is a non-obvious way of, 
from what I can tell, selling only high end memory when e.g. different CAS 
latency is available elsewhere - again, not obvious what their CL is (perhaps 
it is listed somewhere). and maybe other specs apply.

Re: [ccp4bb] Mac mini advice

[Antony Oliver]

Hi Cara, 

Any reason for the Mac Mini over the iMac? - presumably as you've got a 
suitable monitor / keyboard already? 

We're pretty much exclusively iMac of late (ditched the towers) and finding 
them absolutely fine for both fairly intensive jobs (refinement) and 
visualisation/building (Coot / PyMOL). 

We working with the quad-core i7's - but a lower spec is probably ok too - just 
boost the memory if you can. 

With regards, 

Tony. 

Sent from my iPhone

On 22 Jan 2013, at 18:00, Cara Vaughan c.vaug...@mail.cryst.bbk.ac.uk wrote:

 Dear CCP4BB
 
 I'm thinking about buying a Mac Mini and was looking for advice from people 
 who have used these for crystallography.
 
 We don't need the computer to do serious number-crunching as we have back-end 
 servers that can do this for us, so it is primarily for running coot for 
 model building, etc. and low intensity crystallography jobs.
 
 I've seen from the archive that some people do use the Mac Mini for 
 crystallography and I've got two questions:
 1. Do I need the Quad core or is a Dual core processor enough?
 2. Is the intergrated Intel HD graphics card OK for crystallography 
 requirements?
 
 All the best,
 Cara.
 
 
 Cara Vaughan
 Lecturer in Structural Biology
 Institute of Structural and Molecular Biology
 Birkbeck College and UCL
 London UK
 
 
 This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] drawing topology digram

[Antony Oliver]

PDBSUM works equally well for undeposited structures. Hit the generate button 
on the left hand side. 

Tony. 

Sent from my iPhone

On 15 Dec 2012, at 21:04, Wenhua ZHANG xtal.zh...@gmail.com wrote:

 Dear all,
 
  Could anyone recommend me an easy program to draw the secondary topology 
 digram of protein structure? The PDBsum seems working only for deposited 
 structures.
 
  Thank you.
 
 Wenhua ZHANG 

Re: [ccp4bb] [COOT] CCP4 - Coot in Applications

[Antony Oliver]

Thanks Scott.

Using your stand-alone Coot package - 0.7 (revision 4459) [with guile 1.8.8 
embedded] [with python 2.7.3 embedded], everything works perfectly.
The previous issue of invalid window errors no longer occurs, and all scripts 
that use coot now work correctly (as the path /usr/local/bin/coot is defined 
and understood)

I would tentatively suggest therefore that there are some build issues with the 
Coot provided as a package from CCP4 - but *weirdly* only when you invoke it 
from the command line using /Applications/coot.app/Contents/Resources/script - 
which frequently crashes X11…

As a reminder - this was causing me quite a few issues, as other software 
packages don't expect Coot to live in /Applications - and symbolic linking / 
path defining wasn't working consistently.

NB: Running OS X 10.7.5 (Lion).

With regards

Tony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Dec 6, 2012, at 6:05 AM, William G. Scott wrote:

 Dear Tony:
 
 Any chance you might be willing to try installing from here?
 
 http://scottlab.ucsc.edu/~wgscott/xtal/wiki/index.php/Stand-Alone_Coot
 
 That might give us a positive control (if it works) or help to track down the 
 problem (if it doesn't).
 
 It should work the way you want it to.
 
 Bill
 
 
 William G. Scott
 Professor
 Department of Chemistry and Biochemistry
 and The Center for the Molecular Biology of RNA
 228 Sinsheimer Laboratories
 University of California at Santa Cruz
 Santa Cruz, California 95064
 USA



 Ok - annoying thing….
 
 1) using /Applications/coot.app/Contents/Resources/script to launch Coot from 
 the command line (terminal window) works IF you don't have X11 open already.
 2) if you DO have X11 open, coot pretty much always crashes with the 
 aforementioned invalid window errors.
 
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSGetSurfaceBounds
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorFailure: Set a 
 breakpoint @ CGErrorBreakpoint() to catch errors as they are logged.
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 
 Ideas and suggestions welcome.  I guess I could make sure that X11 is not 
 running each time I invoke a script or run coot from the command line - but 
 it's not exactly ideal.
 
 -- NB: Running Mac OS X, 10.7.5 - Lion.

Re: [ccp4bb] [COOT] CCP4 - Coot in Applications

[Antony Oliver]

Thanks Charles - that seems to work..!

Tony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Nov 26, 2012, at 10:19 AM, Charles Ballard wrote:

 You could give
 
 /Applications/coot.app/Contents/Resources/script
 
 a go.  
 
 Charles
 
 On 23 Nov 2012, at 12:20, Antony Oliver wrote:
 
 Forgive what may be a very simple question…
 
 I have recently installed coot using the very nice CCP4 .dmg (0.7.0-i386) - 
 which now puts coot into /Applications
 I now need to set a $PATH to coot, such that various scripts now 'know' 
 where it is (i.e. Phenix and BUSTER - sorry CCP4-ers).
 
 Here I have hit a hurdle.  Which binary file do I need to point a path 
 variable to?
 
 From terminal I can use the command ' open -a coot ' - everything works fine 
 and dandy.
 However, I cannot use ' /Applications/coot.app/Contents/MacOS/coot ' 
 reliably - sometimes it works, but most of the time it crashes with the 
 following errors...
 
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSGetSurfaceBounds
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorFailure: Set a breakpoint 
 @ CGErrorBreakpoint() to catch errors as they are logged.
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
 CGSBindSurface: Invalid window 0x8da
 
 Am I making a school-boy error here?
 
 With many thanks,
 
 Tony. 
 
 
 ---
 Dr Antony W Oliver
 Senior Research Fellow
 CR-UK DNA Repair Enzymes Group
 Genome Damage and Stability Centre
 Science Park Road
 University of Sussex
 Falmer, Brighton, BN1 9RQ
 
 email: antony.oli...@sussex.ac.uk
 tel (office): +44 (0)1273 678349
 tel (lab): +44 (0)1273 677512
 -
 
 
 -- 
 Scanned by iCritical.
 
 

Re: [ccp4bb] CCP4 - Coot in Applications

[Antony Oliver]

Forgive what may be a very simple question…

I have recently installed coot using the very nice CCP4 .dmg (0.7.0-i386) - 
which now puts coot into /Applications
I now need to set a $PATH to coot, such that various home-made scripts now 
'know' where it is 

Here I have hit a hurdle.  Which binary file do I need to point a path variable 
to?

From terminal I can use the command ' open -a coot ' - everything works fine 
and dandy.
However, I cannot use ' /Applications/coot.app/Contents/MacOS/coot ' reliably - 
sometimes it works, but most of the time it crashes with the following errors...

Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
CGSGetSurfaceBounds
Nov 23 12:17:02 coot-real[80005] Error: kCGErrorFailure: Set a breakpoint @ 
CGErrorBreakpoint() to catch errors as they are logged.
Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
CGSBindSurface: Invalid window 0x8da
Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
CGSBindSurface: Invalid window 0x8da
Nov 23 12:17:02 coot-real[80005] Error: kCGErrorIllegalArgument: 
CGSBindSurface: Invalid window 0x8da

coot-real also fails - as it's obviously looking in the wrong place for files…

Acquiring application resources from 
/Users/charlesb/autobuild/Darwin-ras03-006.rl.ac.uk/coot-0.7.0-gtk2-python/share/coot/cootrc
INFO:: splash_screen_pixmap_dir 
/Users/charlesb/autobuild/Darwin-ras03-006.rl.ac.uk/coot-0.7.0-gtk2-python/share/coot/pixmaps


Any ideas or suggestions would be very welcome.


With thanks,

Antony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Nov 23, 2012, at 1:19 PM, Paul Emsley wrote:

[ccp4bb] Copying R-free flags - possibly daft question.

[Antony Oliver]

Am I worrying about something unnecessarily?

I have several protein-drug datasets, all in the same spacegroup, but wildly 
varying resolutions.
I wish to use the same reflections for calculating R-free in all cases.

Using xia2 with a reference dataset for both indexing and R-free seems to work 
fine, apart from the fact that the resulting mtz file, now contains R-free 
labels for reflections that have no observations… i.e. taken from the higher 
resolution reference dataset; see output below.

I get essentially the same results using CAD to copy the R-free column between 
mtzfiles….

Is this actually a problem - or is it just my innate sense of tidiness that 
wants the resolution values to be the same?


Many thanks,

Antony.

 Col SortMinMaxNum  % Mean Mean   Resolution   Type 
Column
 num order   Missing complete  abs.   LowHigh   
label

   1 ASC  0  36  0  100.00 14.2 14.2  57.66   2.02   H  H
   2 NONE 0  36  0  100.00 14.6 14.6  57.66   2.02   H  K
   3 NONE 0  38  0  100.00 14.8 14.8  57.66   2.02   H  L
   4 NONE0.019.0   132   99.18 9.52 9.52  57.40   2.02   I  
FreeR_flag
   5 NONE  -30.6 10855.5 11647   28.03   202.72   203.43  57.66   3.17   J  
IMEAN
   6 NONE1.5   356.4 11647   28.0311.5211.52  57.66   3.17   Q  
SIGIMEAN
   7 NONE7.8  1040.0 11647   28.03   109.05   109.05  57.66   3.17   F  F
   8 NONE1.423.8 11647   28.03 6.59 6.59  57.66   3.17   Q  SIGF


 No. of reflections used in FILE STATISTICS16183





---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] Copying R-free flags - possibly daft question.

[Antony Oliver]

Thanks Eleanor - guess I should have plumbed the depths of the CAD manual a 
little further.

Works perfectly.

Antony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Nov 5, 2012, at 11:24 AM, Eleanor Dodson wrote:

 Just give CAD the resolution cut off of the new data set..
 Eleanor
 From the CAD documentation..
 RESOLUTION [ RESOLUTION OVERALL dmin dmax ] | [RESOLUTION FILE_NUMBER i 
 dmin dmax ]
 
 Use either:
 
 RESOLUTION OVERALL dmin dmax
 for overall resolution limits, or:
 RESOLUTION FILE_NUMBER i dmin dmax
 to set input limit for FILE_NUMBER i.
 dmax, dmin are the resolution limits for the data to be included, i.e. 
 data are included for which 
 (1/dmax)**2 = 4 sin**2theta/lambda**2 =(1/dmin)**2 
 NOTE: Defaults are 0.1 and 1000.0 Angstrom.
 
 
 On 5 Nov 2012, at 09:53, Antony Oliver wrote:
 
 Am I worrying about something unnecessarily?
 
 I have several protein-drug datasets, all in the same spacegroup, but wildly 
 varying resolutions.
 I wish to use the same reflections for calculating R-free in all cases.
 
 Using xia2 with a reference dataset for both indexing and R-free seems to 
 work fine, apart from the fact that the resulting mtz file, now contains 
 R-free labels for reflections that have no observations… i.e. taken from the 
 higher resolution reference dataset; see output below.
 
 I get essentially the same results using CAD to copy the R-free column 
 between mtzfiles….
 
 Is this actually a problem - or is it just my innate sense of tidiness that 
 wants the resolution values to be the same?
 
 
 Many thanks,
 
 Antony.
 
  Col SortMinMaxNum  % Mean Mean   Resolution   Type 
 Column
  num order   Missing complete  abs.   LowHigh   
 label 
 
1 ASC  0  36  0  100.00 14.2 14.2  57.66   2.02   H  H
2 NONE 0  36  0  100.00 14.6 14.6  57.66   2.02   H  K
3 NONE 0  38  0  100.00 14.8 14.8  57.66   2.02   H  L
4 NONE0.019.0   132   99.18 9.52 9.52  57.40   2.02   I  
 FreeR_flag
5 NONE  -30.6 10855.5 11647   28.03   202.72   203.43  57.66   3.17   J  
 IMEAN
6 NONE1.5   356.4 11647   28.0311.5211.52  57.66   3.17   Q  
 SIGIMEAN
7 NONE7.8  1040.0 11647   28.03   109.05   109.05  57.66   3.17   F  F
8 NONE1.423.8 11647   28.03 6.59 6.59  57.66   3.17   Q  
 SIGF
 
 
  No. of reflections used in FILE STATISTICS16183
 
 
 
 
 
 ---
 Dr Antony W Oliver
 Senior Research Fellow
 CR-UK DNA Repair Enzymes Group
 Genome Damage and Stability Centre
 Science Park Road
 University of Sussex
 Falmer, Brighton, BN1 9RQ
 
 email: antony.oli...@sussex.ac.uk
 tel (office): +44 (0)1273 678349
 tel (lab): +44 (0)1273 677512
 
 

Re: [ccp4bb] Coot 0.7 Saving Dialog opens behind main window

[Antony Oliver]

Unfortunately, that very much depends on which OSX you are running (Leopard, 
Snow Leopard, Lion, Mountain Lion) and which keyboard you have…!

On my keyboard it's F3 and not F10. 

T.


---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Oct 31, 2012, at 9:30 AM, Damian Niegowski wrote:

 Choosing one of the desired program/instance windows and pressing F10 is 
 probably the most simple way. F10 will show you all windows open
 accosiated with the one you choose.
 
 
 Damian Niegowski Ph.D.
 Institute of Medical Biochemistry and Biophysics
 Karolinska Institutet
 Scheeles väg 2
 171 77 STOCKHOLM  
 e-mail: damian.niegow...@ki.se
 phone: 0046 8 524 876 33
 fax: 0046 8 736 04 39
 
 On Oct 31, 2012, at 10:13 AM, Miguel Ortiz Lombardía wrote:
 
 Le 30/10/12 20:28, Jason Busby a écrit :
 Another work-around is to use the command-tilde (⌘ + ~) keystroke.  That 
 will cycle through all the windows of the current program.
 
 Jason.
 
 --
 Jason Busby
 PhD Student
 Laboratory of Structural Biology
 School of Biological Sciences
 University of Auckland
 Thomas Building 110
 3a Symonds St
 Private Bag 92019
 Auckland  1142
 New Zealand
 
 ph:  +64 9 3737599 ext 84155
 fx:  +64 9 3737414
 
 On 31/10/2012, at 4:58 AM, Damian Niegowski wrote:
 
 If you choose to use the excellent Mac OSX feature Exposé and Active 
 screen corners this becomes
 less of a problem.
 
 
 Damian Niegowski Ph.D.
 Institute of Medical Biochemistry and Biophysics
 Karolinska Institutet
 Scheeles väg 2
 171 77 STOCKHOLM   
 e-mail: damian.niegow...@ki.se
 phone: 0046 8 524 876 33
 fax: 0046 8 736 04 39
 
 On Oct 30, 2012, at 4:32 PM, Eike Schulz wrote:
 
 Dear Coot-users,
 
 I am running Coot-0.7 on OSX 10.6.8. Installation from the 'Scott'-package
 was no problem at all ­ it runs very smoothly.
 
 However, whenever I want to save the coordinates the saving dialog open
 -behind- the main window. To be more precise: the coordinate molecule
 selector opens in front of it but the -file name selector- opens behind
 the main window. This is over the time a bit frustrating when you have to
 minimize/move the main window every time you want to save your structure.
 
 Does it happen to others as well, or is this specific to my system? If its
 possible, how could it be changed to open in front of the main window?
 
   
   Best regards
 
   Eike
 
 
 
 
 Hi,
 
 I don't know for your osx version (mine is 10.6.8) but the short-cut I
 know is command-grave accent (⌘ + `). One problem with this is that osx
 considers all X11-based applications to be a single X11 application, so
 it can take a while until you reach the window you want if you have
 other X11-based applications open. Another problem is that, contrary to
 what you have in aqua-based applications, the X11 windows don't cycle.
 Apple developers have known about these issues for a long, long time.
 Perhaps they solved them in more recent versions of osx.
 
 Best regards,
 
 -- 
 Miguel
 
 Architecture et Fonction des Macromolécules Biologiques (UMR7257)
 CNRS, Aix-Marseille Université
 Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
 Tel: +33(0) 491 82 55 93
 Fax: +33(0) 491 26 67 20
 mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr
 http://w2.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia
 

Re: [ccp4bb] Coot 0.7 Saving Dialog opens behind main window

[Antony Oliver]

Eike - 

This unfortunately, is a well-known feature of coot on OS X - something to do 
with Apple's implementation of X11.  
Perhaps Paul knows if this happens on Mountain Lion, now that you need to use 
Quartz X11?

Tony.

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

On Oct 30, 2012, at 3:32 PM, Eike Schulz wrote:

 Dear Coot-users,
 
 I am running Coot-0.7 on OSX 10.6.8. Installation from the 'Scott'-package
 was no problem at all ­ it runs very smoothly.
 
 However, whenever I want to save the coordinates the saving dialog open
 -behind- the main window. To be more precise: the coordinate molecule
 selector opens in front of it but the -file name selector- opens behind
 the main window. This is over the time a bit frustrating when you have to
 minimize/move the main window every time you want to save your structure.
 
 Does it happen to others as well, or is this specific to my system? If its
 possible, how could it be changed to open in front of the main window?
 
   
   Best regards
 
   Eike
 
 

[ccp4bb] REMINDER: 3 x Postdoctoral Research Fellow positions at the University of Sussex ( DNA Damage Repair and Signalling)

[Antony Oliver]

- - REMINDER - -
IMMINENT CLOSING DATE: 28th September 2012

3 (Three) Cancer Research UK-funded Postdoctoral Research Fellow positions
are available immediately in the laboratory of Professor Laurence Pearl
FRS and Dr Antony Oliver, to study the structural basis for the assembly
and specificity of multi-protein complexes involved in the recognition and
repair of DNA damage, and DNA damage signalling.

Additional information, plus full details of how to apply for each of the
posts can be found on the University of Sussex website at the following
URL:  http://www.sussex.ac.uk/aboutus/jobs/768

Please follow the application instructions carefully.

The formal closing date for applications is the 28th September 2012.

- - -

The internationally renowned MRC Genome Damage and Stability Centre and
the School of Life Sciences are very well equipped for all aspects of
modern structural biology, with state-of-the-art laboratories for
molecular biology, recombinant expression in bacterial and eukaryotic
hosts, biochemistry, biophysics and X-ray crystallography.

Excellent synchrotron access (~ 2 days/month) is also available through
rolling beam-allocation programmes at both the Diamond Light Source and
ESRF. 

Applicants must have a PhD, and extensive experience in recombinant
expression and protein purification. Previous experience of
crystallization and X-ray crystallography would be a distinct advantage.

The post-holder will be responsible for expression, purification,
crystallization and structure determination of protein-protein and
protein-DNA complexes, plus downstream biochemical and biophysical
characterisation. 

** Informal enquiries only ** may be made to either Professor Pearl [
laurence.pe...@sussex.ac.uk ], or Dr Oliver [ antony.oli...@sussex.ac.uk ].

Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?

[Antony Oliver]

GSH will reduce your protein quite nicely - is your enzyme activity redox
sensitive?  

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512






On 8/29/12 5:56 PM, Peter Hsu hsuu...@u.washington.edu wrote:

Hi all,

I've been purifying my protein off a GST column and have noticed a
massive difference in activity of my protein between a prep that was
freed from the column via on column cleavage, and a prep that was eluted
(20mM GSH) and then cleaved and further purified. I'm suspecting that the
glutathione is somehow modifying/inhibiting my protein in some way,
despite having removed the glutathione from the buffer via dialysis/ion
exchange. I don't see anything out of the ordinary in my electron density
that would suggest that glutathione has affected my protein in some way,
but the huge difference seen in my activity assay suggests otherwise.

My question is, has anyone else seen an effect from glutathione affecting
their protein in some way? My second question is, what's the minimum
amount of glutathione necessary to elute your protein from a column?

Sorry for the off topic question and thanks for any responses,

Peter

Re: [ccp4bb] Various OSes and Crystallography

[Antony Oliver]

Mountain Lion does not come with X11 preinstalled.  However, as Nat
states, you can very easily install Xquartz
Thus far, all of the crystallography programs that were working under Snow
Leopard and Lion are still working on my laptop with Mountain Lion.

-Tony

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512






On 8/9/12 4:25 PM, Nat Echols nathaniel.ech...@gmail.com wrote:

On Thu, Aug 9, 2012 at 8:14 AM, Quentin Delettre q...@hotmail.fr wrote:
 I have seen that in the last Mac Os, X11 have been removed... But can
still
 be used with some package installation.

I guess it isn't distributed with the OS any more - but it is still
available:

http://xquartz.macosforge.org/landing/

-Nat

[ccp4bb] 3 x Postdoctoral Research Fellow positions at the University of Sussex ( DNA Damage Repair and Signalling)

[Antony Oliver]

3 (Three) Cancer Research UK-funded Postdoctoral Research Fellow positions
are available immediately in the laboratory of Professor Laurence Pearl
FRS and Dr Antony Oliver, to study the structural basis for the assembly
and specificity of multi-protein complexes involved in the recognition and
repair of DNA damage, and DNA damage signalling.

Additional information, plus full details of how to apply for each of the
posts can be found on the University of Sussex website at the following
URL:  http://www.sussex.ac.uk/aboutus/jobs/768

The formal closing date for applications is the 28th September 2012.

- - -

The internationally renowned MRC Genome Damage and Stability Centre and
the School of Life Sciences are very well equipped for all aspects of
modern structural biology, with state-of-the-art laboratories for
molecular biology, recombinant expression in bacterial and eukaryotic
hosts, biochemistry, biophysics and X-ray crystallography.

Excellent synchrotron access (~ 2 days/month) is also available through
rolling beam-allocation programmes at both the Diamond Light Source and
ESRF. 

Applicants must have a PhD, and extensive experience in recombinant
expression and protein purification. Previous experience of
crystallization and X-ray crystallography would be a distinct advantage.

The post-holder will be responsible for expression, purification,
crystallization and structure determination of protein-protein and
protein-DNA complexes, plus downstream biochemical and biophysical
characterisation. 

** Informal enquiries only ** may be made to either Professor Pearl [
laurence.pe...@sussex.ac.uk ], or Dr Oliver [ antony.oli...@sussex.ac.uk ].

- - -

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] Enhancing Crystal Quality

[Antony Oliver]

Have you tried different cryoprotectants? Can make a huge difference. Also, 
have you shot an xtal at room temp - to see what the intrinsic diffraction 
limit is? Additive screens? If all else fails you may well need to explore a 
different expression construct. 

Tony. 

Sent from my iPhone

On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote:

 Hi CCP4BBers,
 
 I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM 
 cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave 
 triangle pyramid like crystals. I brought the crystals to synchrotron using 
 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or 
 worse. I have tried changing pH and concentrations of PEG, PEG types. I found 
 out this crystal only grew between pH 6.5~7.5 and PEG types did not change 
 the result of diffraction dramatically. I have also tried the seeding (break 
 it down and reseed in the same condition. Maybe I did it wrong?). It gave me 
 the similar results, not improving. Is there any simple way of improving it 
 before jumping into reengineering the protein.
 
 Thanks,
 
 Lucas

Re: [ccp4bb] NCS rotamers

[Antony Oliver]

forgive the cross-posting coot-bb/ccp4-bb

Can I second that please?  I am possibly in a similar situation -
2.8 Angstrom structure, 6 molecules in the asymmetric unit, refining with
ncs torsion restraint.
It would be very useful to identify which side-chains are in different
rotamers (without having to look at each and every side-chain).

Tony. 

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512




On 6/20/12 10:04 AM, Luca Pellegrini lp...@cam.ac.uk wrote:

Hello,

Is there a way to flag up residues that have been modelled with different
side chain rotamers in two NCS-related molecules? I can use the NCS Ghost
Control tool to check individual residues but it would be useful to be
able to produce a list, so that one can zoom in on possible outliers.

Thanks,
Luca  

Re: [ccp4bb] SUMO(ULP-1) protease

[Antony Oliver]

Gene synthesis and make your own!

Tony. 

Sent from my iPhone

On 24 May 2012, at 20:42, Gloria Borgstahl gborgst...@gmail.com wrote:

 My fellow crystallographers,
 We are thinking the SUMO/His vectors would be nice to have in the lab
 aresenal... but.  The stumbling block is that the protease needed for
 cleavage is very expensive at crystallography scale.  SUMO(ULP-1)
 protease costs ~$700/mg fusion protein.   It would not be a problem
 for labs using micrograms of protein, but is prohibitive at our level
 of protein purification.
 Has anyone found a way around this?  Your pal, Gloria

Re: [ccp4bb] pdb and cif file generation from smiles string

[Antony Oliver]

Considering all the recent posts on this very forum regarding the excellent 
Grade; I would suggest a quick visit here…

http://grade.globalphasing.org/cgi-bin/grade/server.cgi

Tony.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512


From: Shya Biswas shyabis...@gmail.commailto:shyabis...@gmail.com
Reply-To: Shya Biswas shyabis...@gmail.commailto:shyabis...@gmail.com
Date: Wed, 9 May 2012 12:08:39 -0400
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pdb and cif file generation from smiles string

Hi all,
I am having trouble generating a pdb and cif file from the following smiles 
string:
O=C(C[N+]23CN1CN(CN(C1)C2)C3)c45c45

Prodrg fails to run when i draw the molecule in JME editor was wondering if 
anyone knows a better program which does this kind of job.
thanks in advance,
shya

[ccp4bb] Off Topic: Sucrose / Glycerol Gradient Maker Fractionator

[Antony Oliver]

Dear all,

I find myself working on a number of  large multi-protein complexes, and am 
likely to need to use Sucrose / Glycerol gradients in preparing them.
IWhilst I can do this manually in the short term,  I was wondering if someone 
could recommend any manufacturer's (preferably Europe/UK based) that make 
suitable systems for:

  1.  Making the gradients in the first place
  2.  Fractionating the gradients after they have been in the centrifuge.

With a great many thanks,

Tony.


---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] negative density at some places in the side chain of residues

[Antony Oliver]

Alaksa,

1) What rmsd / sigma are you contouring your density at ? i.e. are you down in 
the noise or are you at a reasonable value for your Fo-Fc map?

2) It looks like some of your side-chains appear to have more than one 
conformation - it's fairly easy in Coot to position and model both. 

Tony. 

---
Mobile Account
---

On 26 Apr 2012, at 21:55, Alaksa xtal.cc...@gmail.com wrote:

 
 Dear all
 I am refining the crystal structure of a protein (Rfree and Rvalue are 25 
 and 20 A respectively). However, I am getting the negative density at some 
 places in the side chain of residues. All side chains are properly fitting 
 into the blue density, however red density blobs are also present at the same 
 place along with blue density . At some other place this red density is also 
 present in the main chain along with blue density (see the attached snaps). 
 If I have mutate the residues to alanine then density becomes blue, but when 
 change into the original residue, after refmac5 again it is showing red blob. 
 Also if I rotate the chain to place it in green density, but after running 
 refmac it attain original position having red blob. I am not using TLS.
 I am seeking some strategy so that the problem can be solved. Please suggest 
 me the possible reasons and remedy. Also i am naive in crystallography.
 
 Thanks
 Alaksa
 
 
 coot2.png
 coot1.png
 coot3.png
 coot4.png

Re: [ccp4bb] Arp/WARP for multi-chain complex

[Antony Oliver]

In the absence of a likely, more sensible, answer - I think the trick is/was to 
simply put everything in one pir file, but  link each sequence with a run of 
20 or so alanines i.e. sequence A followed by  ...  sequence B  
    sequence C. 

There may well be a more elegant solution - but I'm fairly sure this worked 
previously for us.  

With regards,

Tony. 


On 19 Apr 2012, at 04:26, Zhou, Tongqing (NIH/VRC) [E] tz...@mail.nih.gov 
wrote:

 Dear All,
 
 I am trying to use Arp/wArp to build an antibody-antigen complex with 1.65 A 
 data, there are three chains (heavy, light chains of antibody and the 
 antigen) in the complex, my question is how to put the sequences in the *.pir 
 file so that it still identifies different chains. It looks like Arp/wArp 
 only accepts *.pir file with one sequence id.
 
 Thanks,
 
 
 Tongqing

Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

[Antony Oliver]

This subject raised (and keeps raising) its head above the parapet not all that 
long ago on this bulletin board.  Maybe it's time to bite the bullet and try 
and do something about it? 

I would like to see and can imagine the following scenario... something I have 
tentatively suggested before...

There is a secure web server (at the PDB?) where you can upload your 
coordinates and structure factor file - a Pre-release server if you will.  On 
uploading you are then given a unique URL which can be provided to a journal 
and passed on to any selected reviewer. Crucially this does *not* allow the 
coordinates or maps to be downloaded, but visually inspected online - via some 
form of web-browser plugin; Aztex Viewer or similar. 

This way reviewers can see the model, and the density that the authors have 
built into, but not have any access to either the coordinate file or mtz - and 
sti make an informed judgment. 

With regards, from a tilting pendolino train, somewhere in the bowels of south 
-east England. 

Tony. 

On 19 Apr 2012, at 14:55, George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de 
wrote:

 Colin,
 
 Speaking as someone who has one foot in small molecule crystallography
 and the other in macromolecular, I have to say that attitudes are
 completely different, and that there are good reasons for this. A PhD
 student or junior postdoc in a macromolecular lab may have spent the
 last three (or more) years cloning, expressing. purifying and
 crystallizing a protein, and it is very likely that three or more groups
 elsewhere in the world are working on the same target. Even if the
 organisms are different, usually only one group will be able to publish
 in a high-profile journal, so being scooped is a major worry and happens
 frequently, even when all concerned are completely honest. A single
 small molecule structure is a very much smaller part of the average
 chemical PhD which often involves dozens of structures, and a couple of
 duplicated structures will have little influence on the future career of
 the PhD student.
 
 Releasing the PDB hold on a structure just before submitting the paper
 has something to be said for it. I would like to do this more often, but
 it is usually vetoed by paranoid biological co-authors. Even if one is
 providing the competition with a good MR model, at least they will have
 to cite it.
 
 George
 
 On 04/19/2012 03:09 PM, Colin Groom wrote:
 It has always struck me as something of a surprise that pre-publication 
 review of structures in protein-land 
 differs so significantly from small molecule-land. One of the activities
 of the CCDC is to supply pre-release
 CSD structures to referees, using a simple, automated system to
 establish that the requestors are referees.
 This avoids the need for any involvement of the depositor or journal and
 allows a centralised record to be kept
 as to who saw which structures and when (although, to my knowledge, we
 have never needed to refer to this).
 In 2012,  requests have averaged at about 5 a day, but the real figure
 is probably much higher, as some journals
 provide this facility themselves. The sense I get from the
 small-molecule community is that they (we) have a
 great degree of well placed trust and see real value in pre-publication
 review of structures, not just papers -
 I'm sure this is true for the overwhelming majority of the
 macromolecular world too.
 
 Colin
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
 herman.schreu...@sanofi.com
 Sent: 19 April 2012 13:54
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
 
 This is off course a valid point. A desperate graduate student faking a
 structure risks his or hers career and reputation, while an anonymous
 referee, borrowing someone else's results gets away without any risk
 of being caught. Besides making the name of the reviewer public, I see
 other options:
 
 1) submit the coordinates and structure factors to the pdb to get a
 priority date as has been suggested before. Many journals require
 anyways a pdb code before acceptance of the paper. One could even
 publish this priority date in the paper in the footnote where the pdb
 code is mentioned.
 2) require from referees a conflict-of-interest-statement that they, or
 close colleagues are not working on the same or a very similar
 structure. If an author gets the impression that he may have been
 scooped by a less-ethical referee, he could ask the journal to verify
 that the referees of his rejected paper were not involved in the
 accelerated publication. If it turns out that a referee has made a false
 statement this would clearly constitute fraud and a reason for
 repercussions.
 
 Herman
 
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Jobichen Chacko
 Sent: Thursday, April 19, 2012 2:12 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Off-topic: 

Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

[Antony Oliver]

Randy,

This is somewhat of a thought experiment at best...but to think on whilst I 
commute home after a great BCA meeting...

You don't necessarily need a password-protected server - just a way of making a 
particularly cryptic URL - which the author is (at first) the only person to 
receive - and able to share a they wish. 

Now that you point out that you could take snapshots and reconstruct the model 
(something that I hadn't quite thought about! )- perhaps having an online 
viewer is a risk, but you'd have to go to quite a bit of effort to steal 
coordinates this way?  Plus you would have to have some diffraction data 
yourself, in order to use the model unfairly to scoop a reviewee...

I certainly appreciate and thank you (!) for the sterling work in producing the 
new validation reports - but as a reviewer myself, it's still not quite the 
same as seeing the maps and fits themselves. 

Tony. 

Sent from my iPhone

On 19 Apr 2012, at 15:39, Randy Read rj...@cam.ac.uk wrote:

 The idea of referees being given a link to the structure at the PDB came up 
 in discussions with PDB people, when we were preparing our X-ray validation 
 report.  Among other potential issues, it would be a lot of work for them to 
 set up a secure password-protected system, and the growth in the PDB keeps 
 them pretty busy doing other things.  The upcoming validation report is meant 
 to satisfy most of what referees would want to know about the structure and 
 its fit to the data.  If it raises some flags, then they have a good excuse 
 to ask for more, through the journal.
 
 On the suggestion of a pre-release server: if you allow someone to rotate a 
 molecule and take several screenshots of it from different orientations, you 
 might as well give them the coordinates because that's all you need to 
 reconstruct them pretty precisely.  For those who know how to compile Fortran 
 programs, Michael Rossmann wrote a program years ago that will extract 
 coordinates from a stereo pair, and I'm sure one could do much better with 
 multiple images.
 
 Regards,
 
 Randy Read
 
 On 19 Apr 2012, at 15:09, Antony Oliver wrote:
 
 This subject raised (and keeps raising) its head above the parapet not all 
 that long ago on this bulletin board.  Maybe it's time to bite the bullet 
 and try and do something about it? 
 
 I would like to see and can imagine the following scenario... something I 
 have tentatively suggested before...
 
 There is a secure web server (at the PDB?) where you can upload your 
 coordinates and structure factor file - a Pre-release server if you will.  
 On uploading you are then given a unique URL which can be provided to a 
 journal and passed on to any selected reviewer. Crucially this does *not* 
 allow the coordinates or maps to be downloaded, but visually inspected 
 online - via some form of web-browser plugin; Aztex Viewer or similar. 
 
 This way reviewers can see the model, and the density that the authors have 
 built into, but not have any access to either the coordinate file or mtz - 
 and sti make an informed judgment. 
 
 With regards, from a tilting pendolino train, somewhere in the bowels of 
 south -east England. 
 
 Tony. 
 
 On 19 Apr 2012, at 14:55, George M. Sheldrick 
 gshe...@shelx.uni-ac.gwdg.de wrote:
 
 Colin,
 
 Speaking as someone who has one foot in small molecule crystallography
 and the other in macromolecular, I have to say that attitudes are
 completely different, and that there are good reasons for this. A PhD
 student or junior postdoc in a macromolecular lab may have spent the
 last three (or more) years cloning, expressing. purifying and
 crystallizing a protein, and it is very likely that three or more groups
 elsewhere in the world are working on the same target. Even if the
 organisms are different, usually only one group will be able to publish
 in a high-profile journal, so being scooped is a major worry and happens
 frequently, even when all concerned are completely honest. A single
 small molecule structure is a very much smaller part of the average
 chemical PhD which often involves dozens of structures, and a couple of
 duplicated structures will have little influence on the future career of
 the PhD student.
 
 Releasing the PDB hold on a structure just before submitting the paper
 has something to be said for it. I would like to do this more often, but
 it is usually vetoed by paranoid biological co-authors. Even if one is
 providing the competition with a good MR model, at least they will have
 to cite it.
 
 George
 
 On 04/19/2012 03:09 PM, Colin Groom wrote:
 It has always struck me as something of a surprise that pre-publication 
 review of structures in protein-land 
 differs so significantly from small molecule-land. One of the activities
 of the CCDC is to supply pre-release
 CSD structures to referees, using a simple, automated system to
 establish that the requestors are referees.
 This avoids the need for any involvement of the depositor or journal

Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

[Antony Oliver]

Thanks Ian, 

Of course it'd have to be something else :-)  but the capability of 
displaying models and maps via a web-browser is at least within current 
capabilities. 

Perhaps the whole model or electron density doesn't need to be presented - 
perhaps just a representative chunk or chunks with the best and worst bits of 
the model and maps (highest / lowest B-factors, Density/Model correlation ? ) 
thereby preventing theft by Fortran Script...?

This is all, of course, just a bit of a thought experiment - and there are 
bound to be problems and issues with such a system - but I think it *is* 
something that could possibly be implemented and would certainly (in my humble 
opinion) be useful for potential reviewers, myself included. 

Tony. 


On 19 Apr 2012, at 15:47, Ian Tickle ianj...@gmail.com wrote:

 Crucially this does *not* allow the coordinates or maps to be downloaded, 
 but visually inspected online - via some form of web-browser plugin; Aztex 
 Viewer or similar.
 
 Anthony, it would have to be something other than AstexViewer since
 the distributed version at least allows you to do a Save As on the
 co-ordinates (not the maps though, but I guess the co-ordinates are
 the main point at issue).
 
 In any case I agree with Randy that there are problems with this.
 
 Cheers
 
 -- Ian

Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

[Antony Oliver]

Tom, that's indeed true. But, the file can be encrypted, and it doesn't 
necessarily have to be in strict PDB format. 

Getting out of my comfort and knowledge zone here... But with the advent of 
HTML5 is a plugin strictly necessary?

Tony. 

Plus if you're only looking at a chunk of structure - you wouldn't have enough 
data to be an issue?


On 19 Apr 2012, at 16:20, Tom Oldfield oldfi...@ebi.ac.uk wrote:

 Hi
 
 I would just like to note that a web-browser plug in is on the client machine 
 - to
 view a PDB file in any viewer like this (ie Astex-viewer, jmol) requires that 
 file to be physically
 downloaded onto the client computer - and put into the TEMP folder of that 
 machine.
 
 As such, the act of viewing the coordinate data in a viewer has by definition 
 downloaded
 the data onto the reviewers computer where they will have complete and 
 unrestricted access
 if they know where to look.
 
 Regards
 Tom
 
 This subject raised (and keeps raising) its head above the parapet not all 
 that long ago on this bulletin board.  Maybe it's time to bite the bullet 
 and try and do something about it?
 
 I would like to see and can imagine the following scenario... something I 
 have tentatively suggested before...
 
 There is a secure web server (at the PDB?) where you can upload your 
 coordinates and structure factor file - a Pre-release server if you will.  
 On uploading you are then given a unique URL which can be provided to a 
 journal and passed on to any selected reviewer. Crucially this does *not* 
 allow the coordinates or maps to be downloaded, but visually inspected 
 online - via some form of web-browser plugin; Aztex Viewer or similar.
 
 This way reviewers can see the model, and the density that the authors have 
 built into, but not have any access to either the coordinate file or mtz - 
 and sti make an informed judgment.
 
 With regards, from a tilting pendolino train, somewhere in the bowels of 
 south -east England.
 
 Tony.
 
 On 19 Apr 2012, at 14:55, George M. 
 Sheldrickgshe...@shelx.uni-ac.gwdg.de  wrote:
 
 Colin,
 
 Speaking as someone who has one foot in small molecule crystallography
 and the other in macromolecular, I have to say that attitudes are
 completely different, and that there are good reasons for this. A PhD
 student or junior postdoc in a macromolecular lab may have spent the
 last three (or more) years cloning, expressing. purifying and
 crystallizing a protein, and it is very likely that three or more groups
 elsewhere in the world are working on the same target. Even if the
 organisms are different, usually only one group will be able to publish
 in a high-profile journal, so being scooped is a major worry and happens
 frequently, even when all concerned are completely honest. A single
 small molecule structure is a very much smaller part of the average
 chemical PhD which often involves dozens of structures, and a couple of
 duplicated structures will have little influence on the future career of
 the PhD student.
 
 Releasing the PDB hold on a structure just before submitting the paper
 has something to be said for it. I would like to do this more often, but
 it is usually vetoed by paranoid biological co-authors. Even if one is
 providing the competition with a good MR model, at least they will have
 to cite it.
 
 George
 
 On 04/19/2012 03:09 PM, Colin Groom wrote:
 It has always struck me as something of a surprise that pre-publication 
 review of structures in protein-land
 differs so significantly from small molecule-land. One of the activities
 of the CCDC is to supply pre-release
 CSD structures to referees, using a simple, automated system to
 establish that the requestors are referees.
 This avoids the need for any involvement of the depositor or journal and
 allows a centralised record to be kept
 as to who saw which structures and when (although, to my knowledge, we
 have never needed to refer to this).
 In 2012,  requests have averaged at about 5 a day, but the real figure
 is probably much higher, as some journals
 provide this facility themselves. The sense I get from the
 small-molecule community is that they (we) have a
 great degree of well placed trust and see real value in pre-publication
 review of structures, not just papers -
 I'm sure this is true for the overwhelming majority of the
 macromolecular world too.
 Colin
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
 herman.schreu...@sanofi.com
 Sent: 19 April 2012 13:54
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
 
 This is off course a valid point. A desperate graduate student faking a
 structure risks his or hers career and reputation, while an anonymous
 referee, borrowing someone else's results gets away without any risk
 of being caught. Besides making the name of the reviewer public, I see
 other options:
 
 1) submit the coordinates and structure factors to the pdb to get a
 

Re: [ccp4bb] Coot chain ID

[Antony Oliver]

Dipankar,

A little bit of cut-and-paste in a good text editor will sort that out fairly 
easily.

Tony.

Sent from my iPhone

On 14 Apr 2012, at 07:54, Dipankar Manna 
dipanka...@aurigene.commailto:dipanka...@aurigene.com wrote:

Dear All,

I fitted a ligand into a structure along with SO4 and waters (COOT). Then I did 
the “Merge Molecules” and did the refinement. In output PDB file the chain ID 
sequence is showing B, A, C( ligand, protein and SO4 respectively) and the ATOM 
numbering is starting from ligand (Chain B instead of Chain A). How can I make 
the sequence A, B and C (Protein, ligand and SO4).

Best wishes

Dipankar



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Re: [ccp4bb] Error in Scala

[Antony Oliver]

One quick thought is - are you trying to scale reflections that don't really 
exist? I.e are you trying to push your resolution a bit too much? 

Tony. 

Sent from my iPhone

On 6 Apr 2012, at 20:29, Yuri Pompeu yuri.pom...@ufl.edu wrote:

 Hi everyone,
 Sorry for the newbie type problems, but I am just starting to use ccp4 for 
 data processing.
 Here is my problem.
 After I index and integrate my images using iMOSFLM, I end up with the .mtz 
 files that contains, AIUI, unmerged reflections.
 Next I should try to merge and scale experimental intensities, using SCALA.
 I am getting an error saying:
  Giving up
 Scala:  Negative Scales 
 task failed.
 
 Anything obvious I am missing? 
 thanks a lot.

Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]

[Antony Oliver]

To my mind it just points to the fact that many scientists are generally
unable to focus on one task or 'thing' at a time.
i.e. very short attention spans...

[before the flamer's start ‹ this is meant as a joke]

Tony.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512






On 4/2/12 9:47 AM, Manfred S. Weiss manfred.we...@helmholtz-berlin.de
wrote:

Dear all,

I find this discussion most amazing. Here, we are dealing with the most
serious issue
that happened to Macromolecular Crystallography since the Alabama case,
and the
whole discussion is centered around singular and plural and Greek and
Latin words
and what not.

In psychology such phenomenon is referred to as displacement activity.

If you are interested, here is the MacMillon definition of it:

http://www.macmillandictionary.com/dictionary/british/displacement-activit
y

Cheers,

Manfred


On 01.04.2012 19:35, Gerard Bricogne wrote:
 On Sun, Apr 01, 2012 at 01:18:15PM -0400, David Schuller wrote:
 On 04/01/12 10:18, Gerard Bricogne wrote:
 Dear Paul,

May I join the mostly silent chorus of Greek/Latin-aware
grumps who
 wince when seeing data treated as singular when it is plural.
 When it are plural?
   Good nit-picking :-) . In my mind the quotes around data would
have
 had the same effect as writing 'the word data', and referring to that
word
 by the 'it'. So there is only one word, while its grammatical number is
 plural.


 At any rate, I heard a Nobel laureate use it incorrectly just two days
ago.
   We shouldn't learn to write by imitating Nobel laureates, then.


   With best wishes,

Gerard.

 --
 ===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu

--
Dr. Manfred. S. Weiss
Helmholtz-Zentrum Berlin für Materialien und Energie
Macromolecular Crystallography (HZB-MX)
Albert-Einstein-Str. 15
D-12489 Berlin
GERMANY
Fon:   +49-30-806213149
Fax:   +49-30-806214975
Web:   http://www.helmholtz-berlin.de/bessy-mx
Email: mswe...@helmholtz-berlin.de




Helmholtz-Zentrum Berlin für Materialien und Energie GmbH

Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher
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Postadresse:
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http://www.helmholtz-berlin.de

Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]

[Antony Oliver]

Think the jury might be out on this one... A quick snip from WikiDictionary...

The plural word phages refers to different types of phage, whereas in common 
usage the word phage can be both singular and plural, referring in the plural 
sense to particles of the same type of phage. Maloy et al: Microbial Genetics, 
2nd ed., 1984

Tony.

---
Mobile Account
---

On 1 Apr 2012, at 16:29, VAN RAAIJ , MARK JOHAN 
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote:

another singular/plural grump:
Recently we can read: phage are.
Phage is singular, the plural is phages (and this does not have that much to do 
with latin or greek).
more reading:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109450/

Quoting Paul Emsley:

The PDBe page for 3k78 says:

The experimental data has been deposited

the data cif file says:

data is under question

Grump.

Is it to late to refer to data as if there were more than one of them?

Anyway, the data mtz file is here if you want to refine with it:

http://lmb.bioch.ox.ac.uk/emsley/data/r3k78sf.mtz

Paul.




Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es

Re: [ccp4bb] contaminant when overexpressing a GST tagged protein

[Antony Oliver]

I would hazard a guess of Gro-EL.

With regards,

Tony.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512


From: SANCHEZ BARRENA, MARIA JOSE 
xmj...@iqfr.csic.esmailto:xmj...@iqfr.csic.es
Reply-To: SANCHEZ BARRENA, MARIA JOSE 
xmj...@iqfr.csic.esmailto:xmj...@iqfr.csic.es
Date: Thu, 22 Mar 2012 16:40:38 +0100
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] contaminant when overexpressing a GST tagged protein

Dear all,

I am trying to express a eukatiotic protein (E. coli codon optimized sequence) 
with a GST tag at the N-terminus. I always get my overexpressed protein and a 
contaminant around 60kDa. This contaminant is not washed out of the column when 
washing glutathione beads with 1M NaCl-buffer. However, during o/n incubation 
with proteases that cleave the GST off (thrombin or TEV), the contaminant is in 
the soluble fraction.

Has someone had this experience? I know about contaminants that bind to Ni2+ 
when overexpressing a His-tagged protein, but this is the first time I get such 
thing with a GST-tagged protein.

One could think that that contaminant could be a protein that binds to my 
overexpressed protein, but I do not think so, cause I always get a huge band of 
the contaminant, independently on the amount of the protein of interest
Many thanks in advance for all your suggestions and sorry for asking about 
non-crystallographic topics.
Regards,

Maria


-
María José Sánchez-Barrena, PhD
Departamento de Cristalografía y Biología Estructural.
Instituto de Química Física Rocasolano. CSIC
Serrano 119. 28006 Madrid (Spain)

Re: [ccp4bb] modelling C-terminal COOH in coot

[Antony Oliver]

There is a button for doing this - add OXT, which if I recall is hidden in the 
other modelling tools menu. 

Tony. 

Sent from my iPhone

On 17 Feb 2012, at 10:26, Hubing Lou louhub...@gmail.com wrote:

 Dear all,
 
 I have a structure solved by molecular replacement. The C-terminus at
 the end of the chain shows as CO, not COOH. How do I make it into COOH
 in COOT? I tried to place an O in the relevant position, but seems not
 working this way.
 
 Thanks,
 
 Hubing

Re: [ccp4bb] DNA length for crystallization

[Antony Oliver]

Lisa, there isn't unfortunately a hard and fast rule for the length of DNA used 
in co-crystallisation. It usually is just a case of screening different 
lengths, permuting the sequence, and investigating overhangs or gaps in the DNA 
duplex.  We generally work with oligos between 8 and 21 nts in length, but 
there are many examples of longer DNAs being co-crystallised, the nucleosome 
comes to mind as an extreme example. 

Do you know if the protein binds better to DNA containing secondary structure 
elements, such as hairpin loops? This can make a difference, especially when 
you don't have sequence-specificity.

Tony O. 

---
Mobile Account
---

On 15 Feb 2012, at 08:07, LISA science...@gmail.com wrote:

 Hi all,
 
 I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA 
 with my protein. But neither of them has good diffraction. Some biochemical 
 data said the longer of DNA, the tigher of the binding betwwen DNA and my 
 protein. The binding is not sequence-specfic. Does anyone have suggestion of 
 the optimization? What is the good length of DNA for crystallization?
 Thank you.
 
 Lisa

Re: [ccp4bb] surface residue mutation

[Antony Oliver]

Have you solved the structure? It's just that you don't say why you need 
different crystal forms.

We had to do a bit of crystal engineering in order to get a complex between 
our protein and a peptide. It turned out to be relatively simple case; visually 
inspecting the crystal packing (in Coot) then mutating, in our case a single 
amino acid, that was generating the crystal lattice.

If you have the structure you could use PISA from the EBI to look for your 
lattice contacts, and choose amino acids there.

If you don't have a structure then things are obviously a bit more complicated. 
You could try and generate homology models (Phyre2) and then mutate surface 
residues, either obvious hydrophobics predicted to point out to solvent, or 
make charge reversal mutants (K to E and vice versa).

Wishing you good luck,

Tony.

Sent from my iPhone

On 14 Feb 2012, at 23:46, Prem Kaushal 
ps...@case.edumailto:ps...@case.edu wrote:


Hi
We have a protein that crystallized in P21212 space group. We are looking for 
some different crystal forms. We tried few things did not work. Now we are 
thinking to mutate surface residues. Anybody aware of any software which can 
predict the mutations that might help in crystallizing protein in different 
space group, please inform me.
Thanks in advance
Prem

--

Re: [ccp4bb] extra density ??

[Antony Oliver]

Stacy, 

It looks like it's just some noise on your two-fold symmetry axis.  
You could probably model some/most of it with a couple of water molecules.

Tony.

On 19 Jan 2012, at 06:44, stacy William wrote:

 Dear All, 
  I am working on plant proteins and solved a structure, there is an extra 
 density which i cannot fix . I am attaching the coot image , can anybody 
 suggest me what it could be
 
 THANKS :)
 coot1.pngcoot2.png

Re: [ccp4bb] Molprobity Clashscore

[Antony Oliver]

Ok, I'm completed baffled... and have obviously started something
unintentionally...

NB: it was a joke! 

I was amused that Molprobity, after 'adding' hydrogens to my model, had
'improved' the clashscore of my model by an obviously unnecessary number
of decimal places...!
[0.0098 point apparently].

Just me apparently.

Off to put my head in a cardboard box.

T.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512






On 1/12/12 11:14 PM, Tim Fenn tim.f...@gmail.com wrote:

On Thu, Jan 12, 2012 at 8:11 AM, Pavel Afonine pafon...@gmail.com wrote:

  Who needs hydrogens?


 may be you need to read this (for example):

 http://www.phenix-online.org/papers/dz5209_reprint.pdf


While this reference is useful, it neglects the role of prior chemical
forces (vdW and electrostatics, for example) in positioning hydrogen
atoms.  The X-ray/neutron data is often not sufficient to uniquely
define an atomic position (hydrogen or otherwise), which can be
especially problematic for atoms with several degrees of freedom, like
water or a hydroxyl hydrogen.  Force fields have come a long way in
defining these forces with reasonable chemical accuracy in the past 10
years, and there is work to show this does benefit X-ray/neutron
refinement (e.g. http://dx.doi.org/10.1016/j.str.2011.01.015) -
suggesting its worthwhile to include this information in X-ray target
functions.  At the very least, it should not be left out of the
discussion, especially when hydrogen atoms are concerned!!!

Regards,
Tim

Re: [ccp4bb] Molprobity Clashscore

[Antony Oliver]

Pavel and CCP4ers.

I did have my tongue firmly in my cheek when mentioning the hydrogens…
I am well aware of their importance [winking smiley]

T.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512


From: Pavel Afonine pafon...@gmail.commailto:pafon...@gmail.com
Date: Thu, 12 Jan 2012 08:11:34 -0800
To: Antony Oliver 
antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
Cc: CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Molprobity Clashscore

Tony,

 Who needs hydrogens?

may be you need to read this (for example):

http://www.phenix-online.org/papers/dz5209_reprint.pdf

?

Pavel

Re: [ccp4bb] Superpose problem

[Antony Oliver]

Dear Tong,

I actually raised this point with the developers of Phaser a while back, at a 
CCP4 meeting, as the wording and nomenclature can be a little confusing at 
times.

Molecular replacement in Phaser can be done with a single structure, or an 
ensemble or group of structures (all of which should be superimposed onto 
each other).

You can also search with more than one ensemble — e.g if your protein comprises 
a number of different domains, say an FHA domain and a kinase domain.

So…

Add superimposed PDB to the ensemble: adds another structure to an exisiting 
group or ensemble, say FHA domains.

Add ensemble, makes a completely new group, to which you can add another set of 
superimposed structures, say kinase domains.

You can then specify a search in Phaser for 2 FHA domains and 1 kinase domain; 
both of which are separate ensembles or groups of structures.

Hopefully this makes some kind of sense?!

Tony.

---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512


From: herohonan heroho...@163.commailto:heroho...@163.com
Reply-To: herohonan heroho...@163.commailto:heroho...@163.com
Date: Wed, 4 Jan 2012 21:16:41 +0800
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Superpose problem

Thank you for your reply!
But I still wonder the differences between Add superimposed PDB file to the 
ensemble and Add ensemble in Phaser program and when to use the former.
If I want to search for a complex model, I often just click the Add ensemble  
till completing the search model.

--
Tong Huo
Ph.D candidate
College of Life Sciences
Nankai University
Tianjin, China


At 2011-12-29 16:56:03,Antony Oliver 
antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk wrote:
You could use the Superpose program to generate 
superpositioned/superposed/superimposed models - i.e. to put your structures 
on top of each other.  There are also a host of other programs available to do 
the same thing (!)

In Phaser, you would then load each of these superimposed models, to generate 
your search ensemble.

The words superposition, superimposition, and superpose, to all intents and 
purposes mean the same thing (ducks behind flame-resistant wall).

With regards, Tony.

Sent from my iPhone

On 29 Dec 2011, at 06:16, SUBSCRIBE CCP4BB Huo tong 
heroho...@163.commailto:heroho...@163.com wrote:

 Hello everyone:
 I am just learning how to use the CCP4 Program suite. When I use the MR 
 approach --Phaser, there is a column say
 Add superimposed PDB file to the ensemble. And there is another CCP4 
 supported program called Superpose.
 I am afraid if my question is professional, but I wonder
 1)if there is any differences between Superpose and Superimpose;
 2)when to use the Superpose or Superimposed function

 Any answer is welcomed!

Re: [ccp4bb] a PDB tool

[Antony Oliver]

You can simply use the renumber residues function in Coot.  Alternatively a 
good text editor, with a replace function can achieve the same result.

Sent from my iPhone

On 3 Jan 2012, at 12:33, Dialing Pretty 
hdc123hdc...@yahoo.commailto:hdc123hdc...@yahoo.com wrote:

Dear All,

I have a PDB file starting from residue 1 to 100 for example, can you introduce 
me a server so that I can convert it to another PDB file starting from 200 to 
300?

Cheers,

Dialing
Newyear.png

Re: [ccp4bb] Superpose problem

[Antony Oliver]

You could use the Superpose program to generate 
superpositioned/superposed/superimposed models - i.e. to put your structures on 
top of each other.  There are also a host of other programs available to do the 
same thing (!)

In Phaser, you would then load each of these superimposed models, to generate 
your search ensemble. 

The words superposition, superimposition, and superpose, to all intents and 
purposes mean the same thing (ducks behind flame-resistant wall). 

With regards, Tony. 

Sent from my iPhone

On 29 Dec 2011, at 06:16, SUBSCRIBE CCP4BB Huo tong heroho...@163.com wrote:

 Hello everyone:
 I am just learning how to use the CCP4 Program suite. When I use the MR 
 approach --Phaser, there is a column say
 Add superimposed PDB file to the ensemble. And there is another CCP4 
 supported program called Superpose.
 I am afraid if my question is professional, but I wonder 
 1)if there is any differences between Superpose and Superimpose;
 2)when to use the Superpose or Superimposed function
 
 Any answer is welcomed!

Re: [ccp4bb] Expression of a Selenomethionine Variant in E. coli

[Antony Oliver]

Generally no, only in the subsequent protein purification steps. 

Tony. 

Sent from my iPhone

On 12 Dec 2011, at 21:57, Yibin Lin yyb...@gmail.com wrote:

 Hi List,
 
 I want to preprare sel-met labeling protein. Could someone can tell me
 if it is necessary to add DTT or BME to medium, which contains
 sel-met, when growing cells?
 
 Thanks in advance,
 
 
 Yibin Lin

[ccp4bb] Bug in XIA2 0.3.3.3 Build 3479

[Antony Oliver]

Dear CCP4 developers,

Forgive the posting here, but I can't find the correct contact details for 
Graeme Winter…

I have just downloaded the latest version of XIA2 (0.3.3.3 build 3479) and 
tried to integrate a dataset.  The program fails with the following error…


Build: 3479
XIA2 0.3.3.3
Command line: xia2 -xinfo process.xinfo -spacegroup I212121
--- Autoindexing SWEEP1 
All possible indexing solutions:
oI  80.58  81.88  82.05  90.00  90.00  90.00
oF  80.56 115.93 116.01  90.00  90.00  90.00
mC  80.56 115.94  70.59  90.00 124.74  90.00
Indexing solution:
oI  80.58  81.88  82.05  90.00  90.00  90.00
 Integrating SWEEP1 
Processed batches 1 to 90
Integration status per image (60/record):
ooo%
%%
o = good% = ok! = bad rmsd
O = overloaded  # = many bad  . = blank
@ = abandoned
Mosaic spread: 0.530  0.660  0.770
Need to rerun the integration...
 Integrating SWEEP1 
Processed batches 1 to 90
Integration status per image (60/record):
ooo%o%o%o%o%
o%
o = good% = ok! = bad rmsd
O = overloaded  # = many bad  . = blank
@ = abandoned
Mosaic spread: 0.520  0.658  0.750
-- Preparing DATA --
Status: error local variable 'reindex_op' referenced before assignment



However, if I run exactly the same command set through an earlier version 
(0.3.3.1 build 3479) I have no problem…


Build: 3479
XIA2 0.3.3.1
Command line: xia2 -xinfo process.xinfo -spacegroup I212121
--- Autoindexing SWEEP1 
All possible indexing solutions:
oI  80.58  81.88  82.05  90.00  90.00  90.00
oF  80.56 115.93 116.01  90.00  90.00  90.00
mC  80.56 115.94  70.59  90.00 124.74  90.00
Indexing solution:
oI  80.58  81.88  82.05  90.00  90.00  90.00
 Integrating SWEEP1 
Processed batches 1 to 90
Integration status per image (60/record):
ooo%
%%
o = good% = ok! = bad rmsd
O = overloaded  # = many bad  . = blank
@ = abandoned
Mosaic spread: 0.530  0.660  0.770
Need to rerun the integration...
 Integrating SWEEP1 
Processed batches 1 to 90
Integration status per image (60/record):
ooo%o%o%o%o%
o%
o = good% = ok! = bad rmsd
O = overloaded  # = many bad  . = blank
@ = abandoned
Mosaic spread: 0.520  0.658  0.750
-- Preparing TNKS --
Likely spacegroups:
I212121
Reindexing to first spacegroup setting: I212121 (h,k,l)

 NB: the program terminates normally.

With regards,

Tony.


---
Dr Antony W Oliver

Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

Re: [ccp4bb] Windows 7 and Xtal Software

[Antony Oliver]

Erm, somewhat confused — if you are going to buy a Mac — why would you need (or 
want!)  a triple boot system? It all seems to work just fine on OS X.

Tony.

On 30 Aug 2011, at 07:38, Nian Huang wrote:

A dual boot laptop is all you need. I always reinstall the windows to get rid 
of bloatware anyway. If you are going to buy a mac, you can also try the triple 
boot, but I don't think anybody is doing it. Although it is very convenient, a 
virtual machine will affect the performance of the software. Nowadays booting 
machine is very fast using a good SSD (under 7 second). So it is really not a 
big trouble comparing before. I am using a sub $400 laptop, and everything runs 
really well under Ubuntu including model building software.

Nian



On Sun, Aug 28, 2011 at 9:23 PM, Jacob Keller 
j-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu wrote:
Dear Crystallographers,

are there any additional problems or known issues running ccp4 or
other xtal software on windows 7 (beyond those of Vista, etc.?) Your
input would be really appreciate before I sink my own personal $$$
into a new laptop

Jacob Keller



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185tel:773.608.9185
email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
***

Re: [ccp4bb] Coot File Save Coordinates

[Antony Oliver]

If you're running Coot on a Mac - it's also unfortunately a well-documented 
feature, something to do with Apple's implementation of X11.


Sent from my iPhone

On 15 Aug 2011, at 21:25, Raji Edayathumangalam 
r...@brandeis.edumailto:r...@brandeis.edu wrote:

Thanks Mischa and Juergen. That was probably my most ridiculous post to the 
CCP4BB!! I found the pop-up dialog box hiding behind all my zillion windows. 
Now why the pop-up window would not pop up actively on top of all other windows 
is a question for another time. Nevertheless, your replies helped :)
Raji


On Mon, Aug 15, 2011 at 3:56 PM, Bosch, Juergen 
mailto:jubo...@jhsph.edujubo...@jhsph.edumailto:jubo...@jhsph.edu wrote:
Have you moved your primary window away ? I mean just in case the pop up window 
opened behind the actual scene window.

Jürgen

On Aug 15, 2011, at 3:54 PM, Raji Edayathumangalam wrote:

Hi Folks,

Apologies for a non-CCP4 question.

I am trying to write our coordinates following SSM superposition using the File 
Save Coordinates option in Coot. But if I click the Select Filename button, 
nothing happens. I thought I would get an option to pick a filename and specify 
what I want my output coordinate filename to be called. But that isn't 
happening. Also, clicking on the molecule on the graphics screen (as someone 
pointed out in a previous post) doesn't help either.

I was able to run this very identical routine several times recently so not 
sure what just happened now! Haven't upgraded Coot or anything. Am using Coot 
0.6.2-pre-1 (revision 3468)  [with guile 1.8.7 embedded] [with python 2.7.1 
embedded].

Help?

Thanks.
Raji

--

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University



..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742tel:%2B1-410-614-4742
Lab:  +1-410-614-4894tel:%2B1-410-614-4894
Fax:  +1-410-955-3655tel:%2B1-410-955-3655
http://web.mac.com/bosch_lab/http://web.mac.com/bosch_lab/






--

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Another paper structure retracted

[Antony Oliver]

PIR is fairly similar to Fasta, from addled memory the format is...

protein name;
empty line
MPREIL...rest of amino acid sequence with an optional asterisk to mark the 
sequence end.

Tony

Sent from my iPhone

On 12 Aug 2011, at 09:14, Phil Evans p...@mrc-lmb.cam.ac.uk wrote:

 Can anyone get this server to work? For me it keeps complaining that my 
 sequence file is not a PIR file. The file looks OK to me, but I've never 
 really understood what a PIR file is
 
 Phil
 
 On 12 Aug 2011, at 01:39, Kevin Jin wrote:
 
 
 Should we really have some crystallographers to review and qc those 
 structures before the formal releasing?  JCSG has set a very good mechanism 
 for this issue.
 
 There is a sever for self check.
 
 http://smb.slac.stanford.edu/jcsg/QC/
 
 
 
 
 
 
 On Thu, Aug 11, 2011 at 4:58 PM, Jacob Keller 
 j-kell...@fsm.northwestern.edu wrote:
 I think they fudged the data in this paper...
 
 JPK
 
 On Thu, Aug 11, 2011 at 6:30 PM, David Schuller dj...@cornell.edu wrote:
 link: http://iai.asm.org/cgi/reprint/IAI.05661-11v1
 
 Ferric C. Fang  Arturo Casadevall
 Retracted Science and the Retraction Index
 Infec. Immun. doi:10.1128/IAI.05661-11
 
 Abstract: Articles may be retracted when their findings are no longer
 considered trustworthy due to scientific misconduct or error, they
 plagiarize previously published work, or are found to violate ethical
 guidelines. Using a novel measure that we call the “retraction index,” we
 found that the frequency of retraction varies among journals and shows a
 strong correlation with the journal impact factor.
 ...
 
 (with special attention to Figure 1, Retraction Index vs. Impact Factor)
 
 
 --
 ===
 All Things Serve the Beam
 ===
  David J. Schuller
  modern man in a post-modern world
  MacCHESS, Cornell University
  schul...@cornell.edu
 
 
 
 
 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 cel: 773.608.9185
 email: j-kell...@northwestern.edu
 ***
 

Re: [ccp4bb] Another paper structure retracted

[Antony Oliver]

Thanks Suku - forgot about the P1, F1 stuff.

Still don't understand why you just can't use the simpler FASTA format with all 
these programs, or even just a plain text file!

Tony.



Sent from my iPhone

On 12 Aug 2011, at 10:18, sukanta mondal 
sukanta.mon...@gmail.commailto:sukanta.mon...@gmail.com wrote:

NBRF/PIR Format:

A sequence in PIR format consists of:

  1.  One line starting with
 *   a  (greater-than) sign, followed by
 *   a two-letter code describing the sequence type (P1, F1, DL, DC, RL, 
RC, or XX), followed by
 *   a semicolon, followed by
 *   the sequence identification code (the database ID-code).
  2.  One line containing a textual description of the sequence.
  3.  One or more lines containing the sequence itself. The end of the sequence 
is marked by a * (asterisk) character

Sequence type - Code

Protein (complete) - P1
Protein (fragment) - F1
DNA (linear) - DL
DNA (circular) - DC
RNA (linear) - RL
RNA (circular) - RC
tRNA - N3
other functional RNA - N1

Example:


P1;CRAB_ANAPL
ALPHA CRYSTALLIN B CHAIN (ALPHA(B)-CRYSTALLIN).
  MDITIHNPLI RRPLFSWLAP SRIFDQIFGE HLQESELLPA SPSLSPFLMR
  SPIFRMPSWL ETGLSEMRLE KDKFSVNLDV KHFSPEELKV KVLGDMVEIH

  GKHEERQDEH GFIAREFNRK YRIPADVDPL TITSSLSLDG VLTVSAPRKQ
  SDVPERSIPI TREEKPAIAG AQRK*

hope this info helps
suku


On Fri, Aug 12, 2011 at 6:11 PM, Phil Evans 
mailto:p...@mrc-lmb.cam.ac.ukp...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk
 wrote:
That's what I had

 protein

GSP etc
...
SEN*


On 12 Aug 2011, at 09:19, Antony Oliver wrote:

 PIR is fairly similar to Fasta, from addled memory the format is...

 protein name;
 empty line
 MPREIL...rest of amino acid sequence with an optional asterisk to mark the 
 sequence end.

 Tony

 Sent from my iPhone

 On 12 Aug 2011, at 09:14, Phil Evans 
 mailto:p...@mrc-lmb.cam.ac.ukp...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk
  wrote:

 Can anyone get this server to work? For me it keeps complaining that my 
 sequence file is not a PIR file. The file looks OK to me, but I've never 
 really understood what a PIR file is

 Phil

 On 12 Aug 2011, at 01:39, Kevin Jin wrote:


 Should we really have some crystallographers to review and qc those 
 structures before the formal releasing?  JCSG has set a very good mechanism 
 for this issue.

 There is a sever for self check.

 http://smb.slac.stanford.edu/jcsg/QC/ 
 http://smb.slac.stanford.edu/jcsg/QC/






 On Thu, Aug 11, 2011 at 4:58 PM, Jacob Keller 
 mailto:j-kell...@fsm.northwestern.eduj-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu
  wrote:
 I think they fudged the data in this paper...

 JPK

 On Thu, Aug 11, 2011 at 6:30 PM, David Schuller 
 mailto:dj...@cornell.edudj...@cornell.edumailto:dj...@cornell.edu 
 wrote:
 link: http://iai.asm.org/cgi/reprint/IAI.05661-11v1 
 http://iai.asm.org/cgi/reprint/IAI.05661-11v1

 Ferric C. Fang  Arturo Casadevall
 Retracted Science and the Retraction Index
 Infec. Immun. doi:10.1128/IAI.05661-11

 Abstract: Articles may be retracted when their findings are no longer
 considered trustworthy due to scientific misconduct or error, they
 plagiarize previously published work, or are found to violate ethical
 guidelines. Using a novel measure that we call the “retraction index,” we
 found that the frequency of retraction varies among journals and shows a
 strong correlation with the journal impact factor.
 ...

 (with special attention to Figure 1, Retraction Index vs. Impact Factor)


 --
 ===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 mailto:schul...@cornell.edu 
 schul...@cornell.edumailto:schul...@cornell.edu




 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 cel: 773.608.9185tel:773.608.9185
 email: mailto:j-kell...@northwestern.edu 
 j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
 ***

Re: [ccp4bb] Another paper structure retracted

[Antony Oliver]

Surely in this ''modern age data could be uploaded to review server whereby 
a reviewer could be given privileged access - to be able to see the model and 
maps, via something like AstexViewer, to gauge the quality and reliability of 
modelling - without actually getting the PDB coordinates or structure factors 
until a manuscript is accepted for publication?

Sent from my iPhone

On 11 Aug 2011, at 11:15, Ethan Merritt merr...@u.washington.edu wrote:

 On Wednesday, 10 August 2011, Nian Huang wrote:
 I Agree with the idea of adding crystallographer reviewers. 
 But accessing to data is not feasible unless there is a good way 
 to protect authors.
 
 Disagree.  
 The data supporting a paper's claims should always be made available
 to the reviewers.  How else can you be assured of a valid review?
 
 The only exception to this I can think of would be human subjects/
 privacy issues, but that must be a rarity in crystallographic papers.
 
Ethan
 
 For
 example, the editor should agree to publish the paper swiftly in advance
 before the data become accessible to reviewers.
 In any case, the flaw of this structure is very clear in the table.
 
 Nian
 
 
 
 On Wed, Aug 10, 2011 at 5:25 PM, Filip Van Petegem 
 filip.vanpete...@gmail.com wrote:
 
 Just another example of where it would have been good for the reviewers to
 get access to the data during the review process...  and where at least one
 of the reviewers *should* be a protein crystallographer...
 
 Filip Van Petegem
 
 On Wed, Aug 10, 2011 at 2:01 PM, David Schuller dj...@cornell.edu wrote:
 
 Time to fuel up the gossip engines for the approaching weekend:
 
 
 http://www.sciencedirect.com/science/article/pii/S096921260800186X
 
 RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound
 to the G-Protein Dimer Gβ1γ2
 Structure, Volume 16, Issue 
 7http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%236269%232008%23999839992%23693753%23FLA%23_cdi=6269_pubType=Jview=c_auth=y_acct=C22719_version=1_urlVersion=0_userid=492137md5=9dc4b8953d3fa243dc98e395b6ac590d,
 9 July 2008, Pages 1086-1094
 Structure 2QNS withdrawn.
 
 --
 ===
 All Things Serve the Beam
 ===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu
 
 
 
 
 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3
 
 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/
 
 

Re: [ccp4bb] Good performing low resolution iterative model building programs?

[Antony Oliver]

Dear Frances, I think the answer is going to somewhat depend on whether you 
need / want de-novo building, i.e straight from independent phases or if you 
have a good quality homology model, that you can use as a reference 
structure, or a good starting point. 

New good things that are available include jelly-roll refinement, and 
refinement utilising information from a reference model - both implemented in 
Phenix and possibly CCP4? 

Tony. 


Sent from my iPhone

On 11 Aug 2011, at 14:31, Francis E Reyes francis.re...@colorado.edu wrote:

 Hi ccp4bb'ers,
 
 Of the automatic model builders out there (autobuild, arp/warp, buccaneer, 
 insert your own here), are there any opinions/personal experience  on which 
 of these perform well in  low resolution cases ( worse than say 3.5-3.8 ) ? 
 
 
 Thanks!
 
 F
 
 -
 Francis E. Reyes M.Sc.
 215 UCB
 University of Colorado at Boulder

Re: [ccp4bb] Another paper structure retracted

[Antony Oliver]

Message below...

Dear Nat,

I think this has actually been implemented, at least on the European side of 
things. I've recently uploaded some structures to the PDB, via the EBI's 
deposition service, and was given a link to the ePDB, with this very useful 
information.  Admittedly, I should have forwarded this to the Journal - but 
didn't. Something to definitely remember for next time I guess.

In response to your question/ideas...

In my mind's eye, this is how it could work - The reviewer's view would be 
available directly from the PDB, so that you are only depositing data once, and 
making thing's that bit more secure  -  a unique URL could then be given to the 
depositor, who would then forward this onto to the journal where they want 
their paper to be published.   Something along the lines of a Kinemage would 
work well I guess, so a judgment could be made as to how well the model fits 
the electron density, by a knowledgable crystallographic reviewer



Sent from my iPhone

On 11 Aug 2011, at 15:03, Nat Echols 
mailto:nathaniel.ech...@gmail.comnathaniel.ech...@gmail.commailto:nathaniel.ech...@gmail.com
 wrote:

2011/8/11 Antony Oliver 
mailto:antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.ukantony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
Surely in this ''modern age data could be uploaded to review server whereby 
a reviewer could be given privileged access - to be able to see the model and 
maps, via something like AstexViewer, to gauge the quality and reliability of 
modelling - without actually getting the PDB coordinates or structure factors 
until a manuscript is accepted for publication?

Wasn't part of the point of the PDB's Validation Task Force to design a 
comprehensive validation report that could be made accessible to reviewers, 
which would capture as many problems as possible with the model and data?  If 
you pull together all of the quality measures from the various programs 
currently in use, it would have made many of the issues with this structure 
very obvious without needing to view the structure itself.  (I like your idea, 
but I think it's actually fairly difficult to keep the data private - although 
converting to an intermediate format like the Molprobity kinemages would be a 
partial solution.)

-Nat

Re: [ccp4bb] prescission protease cutting site

[Antony Oliver]

Dear Jerry,

Our in-house series of vectors encode the Rhinovirus 3C-protease site, followed 
directly by a NdeI site, which after cleavage leaves just GPHM on the front of 
your protein.  We routinely get 100% cleavage with incubation overnight at 4˚C 
— and have several xtal structures to boot.

Best wishes,

Antony.


On 2 Aug 2011, at 17:03, Marco Lolicato wrote:

  We are going to use the prescission protease cutting site (LEVLFQ/GP 
)  in our cloning vector to remove the His6 tag.

  Do we need to insert some linkers between this cutting site and the 
target protein to improve the cleavage efficiency?

  Or we are over concerned.

 Thanks a lot and have a nice summer.

Jerry McCully

Re: [ccp4bb] Phaser and Molrep gave different solutions

[Antony Oliver]

Have you tried using the DNA as your search model? - I have had success this 
way round - certainly more phasing power than your protein model, I guess.  
Also, refine with your DNA in place, and your phases/ map should improve - 
hopefully allowing you to place your protein molecules with ease.

Tony.

Sent from my iPhone

On 21 Jul 2011, at 12:40, Hubing Lou 
louhub...@gmail.commailto:louhub...@gmail.com wrote:

I was worried as well with the low TFZ score. Usually successful cases with 
score 8. I am still puzzled why Phaser and Molrep gave different solutions. 
Does this mean molecular replacement do not work out in this case so more 
crystals have to be prepared?

A little more information might be helpful to dissolve the problem here. The 
model I used is a protein-DNA complex. The protein was Chainsaw editted but the 
DNA sequence was directly borrowed from the original model.

Best,
Hubing

On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic 
mailto:frederic.velli...@ibs.frfrederic.velli...@ibs.frmailto:frederic.velli...@ibs.fr
 wrote:
Hi,

It's not a bad idea to read the Phaser manual for molecular replacement; see 
http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement 
http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement

Soon after the start, in a table on the right hand side, there is: TFZ score  
5, have I solved it ? No.

Hence with a TFZ score of 3.8 you do not have a solution using Phaser.

Fred.

Hubing Lou wrote:
Dear all,

I am stuck in a molecular replacement case and looking for advices.
I have been working on a protein-DNA complex structure.
Data was processed by HKL2000 to 2.6Ang and some of the data statistics are 
shown below:

Space group: P21,
Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90
Redundancy: 2.8 (2.7)
Completeness: 94.8 (93.1)
Linear R-fac: 0.051 (0.442)

Data quality was checked by Phenix.xtriage and there's no problem. I then 
prepared a model by Chainsaw. Our protein shares only 30% of sequence 
similarity with the model, but structurally they are in the same group and 
almost identical in apo form. Matthrews Coeff indaced two monomers in AU. I 
then ran Phaser in automated search mode and there's a solution with RFZ 
score 4.8, TFZ score 3.8. The electron density map was not bad with DNA double 
helix clearly seen. However Refmac5 couldn't get Rfree lower than 50%.

I then changed to MolRep, ran self rotation function first then used the 
first 10 peaks for translation search. Again there's a solution but it is 
different from that from Phaser. I attached a picture here. Checking in coot, 
the packing is the same. But, the refinement couldn't get Rfree lower than 50%.

I have tried to include NCS, TLS refinement in Refmac, both not working.
Hope someone out there can help.
Thanks very much for your time.

Hubing

Re: [ccp4bb] Concentrating a protein solution - subbu

[Antony Oliver]

You could try loading a small 1ml HiTrap (or similar) Q or S column with your 
protein - and knocking it off it in one go with high salt, alternatively 
micro-dialysis against a solution containing PEGs can also work well. 

Tony

Sent from my iPhone

On 21 Jul 2011, at 17:54, Narayanan Ramasubbu ramas...@umdnj.edu wrote:

 Dear All:
 We have been trying to crystallize a protein which is large -  100 kDa. This 
 is soluble but the best we can get is about 1 mg/mL.
 It did crystallize but did not diffract well. Efforts to increase the 
 concentration has been unsuccessful. I am wondering whether there are methods 
 that others use to increase the concentration other that using amicon columns.
 Any help will be appreciated.
 Thanks
 Subbu

[ccp4bb] Postdoctoral Research Fellow - University of Sussex - REVISED CLOSING DATE

[Antony Oliver]

*PLEASE NOTE REVISED CLOSING DATE*

[ Postdoctoral Research Fellow - Genome Damage and Stability Centre, University 
of Sussex - Ref 292 ]


An MRC-funded post is available in the laboratory of Professor Laurence Pearl 
and Dr Antony Oliver to study the structure and function of the Smc5/6 complex. 
 This project forms part of a coordinated multi-disciplinary approach in 
collaboration with the laboratories of Dr Jo Murray, Dr Felicity Watts and Prof 
Alan Lehmann.

The Genome Damage and Stability Centre is an internationally renowned institute 
carrying out research on the response of cells to DNA damage, genome 
instability and its relationship to human disease. It provides a concentration 
of relevant expertise and technologies for cancer research.

We are seeking to appoint a structural biologist to be responsible for 
expression, purification, crystallization and structural analysis of proteins 
that comprise the Smc5/6 complex. Applicants must have a PhD, and experience in 
recombinant expression and protein purification.  Previous experience of 
crystallisation and X-ray crystallography would be an advantage.

The post is full-time, for a fixed term for 36 months, with an expected salary 
range of between £29,972-£35,788 per annum.

*Informal* enquiries about the post can be made to either Professor Pearl; 
laurence.pe...@sussex.ac.ukmailto:laurence.pe...@sussex.ac.uk, or Dr Oliver; 
antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk.


Application forms, plus additional details about the post, can be found at:  
http://www.sussex.ac.uk/Units/staffing/personnl/vacs/vac292-293.shtml

*** Closing date for applications is:  29 July 2011 ***


The University of Sussex is committed to equality of opportunity and we 
encourage diversity in the workplace. We have a legal responsibility to ensure 
that all employees have the right to live and work in the UK. For some 
vacancies we may be able to apply for a Certificate of Sponsorship subject to 
the resident labour market test being met and the preferred candidate being 
able to pass the points-based assessment. Before applying for any jobs please 
check current immigration laws in relation to your circumstances at 
www.ukba.homeoffice.gov.ukhttp://www.ukba.homeoffice.gov.uk

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

[ccp4bb] Postdoctoral Research Fellow - Genome Damage and Stability Centre, University of Sussex - Ref 292

[Antony Oliver]

[ Postdoctoral Research Fellow - Genome Damage and Stability Centre, University 
of Sussex - Ref 292 ]

An MRC-funded post is available in the laboratory of Professor Laurence Pearl 
and Dr Antony Oliver to study the structure and function of the Smc5/6 complex. 
 This project forms part of a coordinated multi-disciplinary approach  in 
collaboration with the laboratories of Dr Jo Murray, Dr Felicity Watts and Prof 
Alan Lehmann.  

The Genome Damage and Stability Centre is an internationally renowned institute 
carrying out research on the response of cells to DNA damage, genome 
instability and its relationship to human disease. It provides a concentration 
of relevant expertise and technologies for cancer research.

We are seeking to appoint a structural biologist to be responsible for 
expression, purification, crystallization and structural analysis of proteins 
that comprise the Smc5/6 complex. Applicants must have a PhD, and experience in 
recombinant expression and protein purification.  Previous experience of 
crystallisation and X-ray crystallography would be an advantage.

The post is full-time, for a fixed term for 36 months, with an expected salary 
range of between £29,972-£35,788 per annum.

Informal enquiries about the post can be made to either Professor Pearl; 
laurence.pe...@sussex.ac.uk, or Dr Oliver; antony.oli...@sussex.ac.uk.

Application forms, plus additional details about the post, can be found at:  
http://www.sussex.ac.uk/Units/staffing/personnl/vacs/vac292-293.shtml

Closing date for applications is:  30 September 2011

The University of Sussex is committed to equality of opportunity and we 
encourage diversity in the workplace. We have a legal responsibility to ensure 
that all employees have the right to live and work in the UK. For some 
vacancies we may be able to apply for a Certificate of Sponsorship subject to 
the resident labour market test being met and the preferred candidate being 
able to pass the points-based assessment. Before applying for any jobs please 
check current immigration laws in relation to your circumstances at 
www.ukba.homeoffice.gov.uk

---
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ

email: antony.oli...@sussex.ac.uk
tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

[ccp4bb] Joint PhD position at the University of Sussex

[Antony Oliver]

UNIVERSITY OF SUSSEX

An MRC-funded PhD position is available immediately in the Genome Damage and 
Stability Centre, School of Life Sciences, University of Sussex.  The student 
will be supervised jointly by the laboratories of Professor Keith Caldecott and 
Dr Antony Oliver, and will experimentally address fundamental questions 
concerning the role of two human DNA repair proteins (XRCC1 and TDP2) that are 
critical for the maintenance of genome stability and impact on neuroprotection 
and cancer. 

The project is divided into two distinct but complementary sub-projects.  For 
one part of the project the student will employ a variety of molecular, 
biochemical, and cellular approaches to identify novel protein partner/s of the 
XRCC1 protein, focusing initially on specific candidates involved in DNA damage 
sensing, signaling, and DNA repair. In addition to this targeted approach, 
affinity-purification and mass spectrometric analysis of epitope-tagged XRCC1 
protein complexes will identify unanticipated novel partners. 

For the second part of the project, the student will address the structure of a 
second novel double-strand break repair protein we have recently identified, 
denoted TDP2 This protein is implicated in the repair of DNA damage induced by 
an important class of chemotherapeutic agents and is a putative target for 
novel anti-cancer agents. 

This PhD project will provide an attractive balance of biochemical, 
biophysical/structural, and cellular approaches, providing the successful 
applicant with a broad and competitive skill-set.

The internationally renowned Genome Damage and Stability Centre and the School 
of Life Sciences is very well equipped for all aspects of modern structural 
biology, with state-of-the-art laboratories for molecular biology, recombinant 
expression in bacterial and eukaryotic systems, biochemistry, biophysics and 
X-ray crystallography.

Informal enquiries can be made to Professor Caldecott 
(k.w.caldec...@sussex.ac.uk) or Dr Oliver (antony.oli...@sussex.ac.uk).

Closing date:  June 24, 2011

For full details and how to apply see:  www.sussex.ac.uk/gdsc/1-1.php

The University of Sussex is committed to equality of opportunity

[ccp4bb] PhD position at the Genome Damage and Stability Centre, University of Sussex

[Antony Oliver]

UNIVERSITY OF SUSSEX

An MRC-funded PhD position is available immediately in the Genome Damage and 
Stability Centre, School of Life Sciences, University of Sussex.  The student 
will be supervised jointly by the laboratories of Professor Keith Caldecott and 
Dr Antony Oliver, and will experimentally address fundamental questions 
concerning the role of two human DNA repair proteins (XRCC1 and TDP2) that are 
critical for the maintenance of genome stability and impact on neuroprotection 
and cancer. 

The project is divided into two distinct but complementary sub-projects.  For 
one part of the project the student will employ a variety of molecular, 
biochemical, and cellular approaches to identify novel protein partner/s of the 
XRCC1 protein, focusing initially on specific candidates involved in DNA damage 
sensing, signaling, and DNA repair. In addition to this targeted approach, 
affinity-purification and mass spectrometric analysis of epitope-tagged XRCC1 
protein complexes will identify unanticipated novel partners. 

For the second part of the project, the student will address the structure of a 
second novel double-strand break repair protein we have recently identified, 
denoted TDP2 This protein is implicated in the repair of DNA damage induced by 
an important class of chemotherapeutic agents and is a putative target for 
novel anti-cancer agents. 

This PhD project will provide an attractive balance of biochemical, 
biophysical/structural, and cellular approaches, providing the successful 
applicant with a broad and competitive skill-set.

The internationally renowned Genome Damage and Stability Centre and the School 
of Life Sciences is very well equipped for all aspects of modern structural 
biology, with state-of-the-art laboratories for molecular biology, recombinant 
expression in bacterial and eukaryotic systems, biochemistry, biophysics and 
X-ray crystallography.

Informal enquiries can be made to Professor Caldecott 
(k.w.caldec...@sussex.ac.uk) or Dr Oliver (antony.oli...@sussex.ac.uk).

Closing date:  June 24, 2011

For full details and how to apply see:  www.sussex.ac.uk/gdsc/1-1.php

The University of Sussex is committed to equality of opportunity

Re: [ccp4bb] Preparation of seed-stocks without seed-beads

[Antony Oliver]

You can melt the end of a thin glass capillary, forming a glass 'bead' at one 
end.
You can then attack your drop, containing the crystals to made into seeds, with 
the beaded end of the capillary.
You can then pipette up the crushed crystals, and dilute and/or spin to make 
your seed stock.

With regards,
Tony.
  
 Dear CCP4bbers,

 Can anyone suggests how to make seed-stocks if one is not having
 seed-beads... Is there any other methods to crush the crystals for the
 same
 purpose. What if it is simple vortexed. Off-course there wold be all sorts
 of sizes, the intact crystals as well.
 Please suggest.

 Thanks a lot more for previous help.
 James.



Dr Antony W. Oliver
Senior Scientist
Cancer Research UK DNA Repair Enzymes Group
Section of Structural Biology
The Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB

antony.oli...@icr.ac.uk
020 7153 5571


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[ccp4bb] Off-topic: Carba-NAD

[Antony Oliver]

Dear bulletin-boarders...

Does anyone know of a good (UK?) source, where I can buy some carba-NAD?
I would like to use it in some co-crystallisation experiments.
I've looked in the 'usual' places - i.e. Sigma-Aldrich, but can't seem to find 
it anywhere.

Many thanks in advance,

Antony

Re: [ccp4bb] Protein-DNA complex prepartion for crystallization

[Antony Oliver]

Dilute both the Protein and DNA before mixing them at the molar ratio 
you require - I would aim to have the protein component at around 1 mg/ml.

Mix, then concentrate together, till you reach the concentration you want.

Antony.

On Wed, Oct 1, 2008 at 6:52 PM, E rajakumar [EMAIL PROTECTED] 
mailto:[EMAIL PROTECTED] wrote:


Dear All
Sorry for non-crystallography query. I am working on
DNA binding protein, while mixing DNA with protein for
preparing Protein-DNA complex for crystallization,
protein is precipitating.  pI of the protein is 9.3
and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol.
Concentration of the protein used for mixing with DNA
is 8 mg/mL. DNA to protein molar ratio is 1.2.  Please
advise me how to prevent precipitation. Is changing of
buffer pH and adding divalent cation like MgCl2 can
help in preventing precipitation?
Thanking You
Rajakumara


E. Rajakumara
Postdoctoral Fellow
 Strcutural Biology Program
 Memorial Sloan-Kettering Cancer Center
 New York-10021





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