Re: [ccp4bb] how to use electron scattering factors in REFMAC program?

[Roberto Steiner]

Hi Benedetta

Use an external keyword file that with the line
'source ec mb’

Best
R


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
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On 9 Feb 2024, at 10:30, BENEDETTA CARROZZINI 
mailto:benedetta.carrozz...@cnr.it>> wrote:

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Dear CCP4BB community,
I am working on the structural solution of macromolecules via MR using 3D 
electron diffraction data.

I would like to know if it is possible to carry out a preliminary refinement 
(under 'kinematic' conditions) of the MR model with REFMAC.

 If so, what are the options (or rather instructions) I should provide to the 
program to correctly use electron scattering factors (instead of the standard 
X-ray ones)?
 I have searched for this information in the manual and online without success 
and for this reason, I ask for your precious help.

Thanks in advance
Dedi
​


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Re: [ccp4bb] Crystallizing a tough target

[Roberto Steiner]

HOD crystallised at 150mg/ml
https://www.pnas.org/doi/10.1073/pnas.0909033107

Once I set the wrong time on the centrifuge and it went up to >200 mg/ml. I am 
sure many others have similar stories.

All the best
Roberto


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
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On 5 Feb 2024, at 11:27, kavyashreem 
mailto:kavyashr...@instem.res.in>> wrote:

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Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




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Re: [ccp4bb] alternatives to Consurf server

[Roberto Steiner]

ConservFold from the Rodrigues lab at Warwick is something that you might 
consider.
https://www.rodrigueslab.com/resources



Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
Viale G. Colombo 3
35131 Padova, Italia
Telefono 0039 049 8276409

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On 4 Jan 2024, at 14:58, Andre Godoy 
<0b9d7671a1b6-dmarc-requ...@jiscmail.ac.uk>
 wrote:

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Dear CCP4ers,

Wishing you all a Happy New Year!

You may have noticed that the Consurf server has been down since the conflicts 
in the Middle East began. Does anyone know of alternative tools that can 
provide similar results?

Best regards.


Andre S. Godoy, PhD

Universidade de São Paulo
Instituto de Física de São Carlos
Av. João Dagnone, 1100, Jd. Santa Angelina
13563-120 - São Carlos, SP, Brazil



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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Roberto Steiner]

Difficult to be sure from a single angle pic but I would suggest Cys oxidation.

There is an important 20 years old paper (see below) on Cys oxidation in a 
tyrosine phosphatase (like yours)

Best
Roberti]


Nature

  *   

  *   

  *   

. 2003 Jun 12;423(6941):773-7.
 doi: 10.1038/nature01681.
Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B
Rob L M van 
Montfort
 1, Miles 
Congreve, 
Dominic Tisi, 
Robin Carr, 
Harren Jhoti


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
Viale G. Colombo 3
35131 Padova, Italia
Telefono 0039 049 8276409

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On 18 Dec 2023, at 18:03, Liliana Margent 
mailto:lmarg...@gradcenter.cuny.edu>> wrote:

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this is important
Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc 
density from a few cysteines during the refinement process. We were wondering 
if anyone had seen something similar so they could offer some insight on the 
likely chemistry at hand, and a potential refinement solution. Attached are two 
images of the observed extra density at two cysteines, 505 and 518. We have 
modeled acetylated cysteine, s-hydroxycysteine, and s-mercaptocysteine but it 
does not solve the density. The protein in question is a Protein Tyrosine 
Phosphatase known as STEP (PTPN5), with data collected to a resolution of 1.79 
Å. The crystals were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of 
note, prior to data collection the crystal was conserved at room temp for long 
time where it dried, and was subsequently rehydrated with mother liquor. Thank 
you so much.


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Re: [ccp4bb] solvent mask for partial ligands

[Roberto Steiner]

Dear Gottfried

Assuming your modelling is correct you can always try occupancy refinement of 
your ligand (or simply test alternative values). Maybe that will help.
You can also play with bulk solvent mask parameters. Keywords (both occupancy 
and mask parameters) can be found in
https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#id.b236767394ac

Best wishes
Roberto


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
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On 14 Dec 2023, at 11:20, Palm, Gottfried 
>
 wrote:

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Dear all,
  I have a modelling problem, which probably has something to do with the 
solvent mask.
There is a positive density blob at the surface of my protein, which I can more 
or less satisfy with a Hepe (EPE). Hepes is in the buffer, the sulfonic acid 
H-bonds to a lysine side chain and a main chain N, one of the ring nitrogens to 
a glutamate side chain. At occupancy 1.0 I get B-factors twice as high as the 
protein and negative difference density. Thus, I set the occupancy to 0.5 and 
get the whole EPE covered in positive difference density at 3 sig, peaking at 6 
sig. This positive density is much larger than without EPE. On first glance, 
this looked contradictory to me: more positive density after putting more atoms 
there. I can only guess that the solvent at the place of the EPE is set to 0 
(instead of 0.5), so I get positive density because of only 0.5 occupied EPE. 
Can I refine with half a ligand and half solvent?
Greetings
  Gottfried


On Tuesday, 12-12-2023 at 09:50 Krieger, James M wrote:
There is also discussion about this for chaperonins such as CCT and GroEL. It 
may be that beryllium fluoride works better.

Best wishes
James

> On 12 Dec 2023, at 05:30, Matthew BOWLER 
> mailto:mbow...@embl.fr>> wrote:
>
> Dear Jinyu,
> we observed exactly this in PGK - see https://doi.org/10.1021/ja100974t where 
> we confirmed it by both crystallography and 19F NMR
>
> In fact ALF3 does not exist as a trigonal bipyramid - all cases where this 
> geometry is observed are likely MgF3 that does adopt this geometry and will 
> replace the aluminium at pH values above 8 as Al will precipitate as AlOH.
>
> We only saw the water replacing the F when a catalytic residue charge was 
> removed and proposed the hypothesis that phos transfer enzymes need to 
> balance the charge in the active site exactly therefore selecting the neutral 
> AlF3 over negative AlF4- that both exist in solution.
>
> Best wishes, Matt
>
>> On 2023-12-12 03:40, Friday wrote:
>> Dear CCP4 community,
>> I recently tried to use AlF3 to mimic the transition of an enzyme. In
>> all the publications I can find, when mimicking the 3rd phosphate of
>> the transition state, the AlF3 forms a trigonal bipyramid
>> coordination, and the G(A)DP oxygen to the AL  is about equal distance
>> to that from the AL to the catalytic water oxygen (2.2A).
>> However, I got a completely different geometry of ALF3, I think I have
>> the ALF3OH bound and it's a  tetrahedron geometry instead of trigonal
>> pyramidal. The G(A)DP oxygen to the AL  is about 3.1A and  the AL to
>> the catalytic water oxygen is about 1.8A.
>> I am pretty sure it is not the ALF4 but the ALF3OH since the AL-OH
>> distance is longer than the Al-F distance (1.8A verse 1.6A) and I have
>> a very high resolution structure.
>> Can anyone explain this to me, have you ever encountered this before?
>> Your answer is much appreciated!
>> Jinyu
>> -
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> --
> Matthew Bowler
> Project Leader MASSIF1@ESRF
> European Molecular Biology Laboratory
> 71 avenue des Martyrs
> CS 90181 F-38042 Grenoble
> France
> ===
> Tel: +33 (0) 4.76.20.76.37
> Fax: +33 (0) 4.76.20.71.99
>
> http://www.embl.fr/
> http://www.esrf.eu/MASSIF1
> https://twitter.com/id30_massif1
> https://www.embl.org/people/person/mbowler/
> ===
>
> 

Re: [ccp4bb] The experiment is still very much needed (though AlphaFold helps a lot)

[Roberto Steiner]

Based however on an AlphaFold version that I guess will be at some point 
released. I was wondering if someone had an early access….
Oh well…I guess we will find out how it performs on ligands soon enough.

Cheers
Roberto


Roberto A Steiner
www.steinerlab.org<http://www.steinerlab.org/>
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk<mailto:roberto.stei...@kcl.ac.uk>
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it<mailto:roberto.stei...@unipd.it>
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
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On 1 Dec 2023, at 14:35, Frank von Delft 
mailto:frank.vonde...@cmd.ox.ac.uk>> wrote:

That's a press release, not a tool that can be tested.


On 01/12/2023 12:19, Roberto Steiner wrote:
Great paper indeed!

However quite significant progress seems to have been achieved with ligands as 
well (and not only)…
 
https://deepmind.google/discover/blog/a-glimpse-of-the-next-generation-of-alphafold/
Has anyone in the community put this to the test?


Best wishes
Roberto


Roberto A Steiner
www.steinerlab.org<http://www.steinerlab.org/>
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk<mailto:roberto.stei...@kcl.ac.uk>
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it<mailto:roberto.stei...@unipd.it>
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
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35131 Padova, Italia
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On 30 Nov 2023, at 22:35, Tom Terwilliger 
<b6116340b489-dmarc-requ...@jiscmail.ac.uk<mailto:b6116340b489-dmarc-requ...@jiscmail.ac.uk>>
 wrote:


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b6116340b489-dmarc-requ...@jiscmail.ac.uk<mailto:b6116340b489-dmarc-requ...@jiscmail.ac.uk>.
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Hi Structural biologist colleagues!

Our article that helps you make the case that the experiment is still very much 
needed is now out:

https://www.nature.com/articles/s41592-023-02087-4

"AlphaFold predictions are valuable hypotheses and accelerate but do not 
replace experimental structure determination."  Nature Methods (2023)

Also, here is a video on the Phenix Tutorials YouTube channel that describes 
this analysis:  https://www.youtube.com/watch?v=ugMPYdPo8Bc

(I hope you will be happy to see that you and your structural biology 
colleagues get the credit for making AlphaFold possible at 2:35 in the 
introduction of this video!  Keep up the fantastic structural biology work!)

All the best,
Tom T
--
Thomas C Terwilliger
Laboratory Fellow, Los Alamos National Laboratory
Senior Scientist, New Mexico Consortium
100 Entrada Dr, Los Alamos, NM 87544
Email: 
tterwilli...@newmexicoconsortium.org<mailto:tterwilli...@newmexicoconsortium.org>
Tel: 505-431-0010




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Re: [ccp4bb] The experiment is still very much needed (though AlphaFold helps a lot)

[Roberto Steiner]

Great paper indeed!

However quite significant progress seems to have been achieved with ligands as 
well (and not only)…
 
https://deepmind.google/discover/blog/a-glimpse-of-the-next-generation-of-alphafold/
Has anyone in the community put this to the test?


Best wishes
Roberto


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
Viale G. Colombo 3
35131 Padova, Italia
Telefono 0039 049 8276409

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On 30 Nov 2023, at 22:35, Tom Terwilliger 
>
 wrote:

You don't often get email from 
b6116340b489-dmarc-requ...@jiscmail.ac.uk.
 Learn why this is important
Hi Structural biologist colleagues!

Our article that helps you make the case that the experiment is still very much 
needed is now out:

https://www.nature.com/articles/s41592-023-02087-4

"AlphaFold predictions are valuable hypotheses and accelerate but do not 
replace experimental structure determination."  Nature Methods (2023)

Also, here is a video on the Phenix Tutorials YouTube channel that describes 
this analysis:  https://www.youtube.com/watch?v=ugMPYdPo8Bc

(I hope you will be happy to see that you and your structural biology 
colleagues get the credit for making AlphaFold possible at 2:35 in the 
introduction of this video!  Keep up the fantastic structural biology work!)

All the best,
Tom T
--
Thomas C Terwilliger
Laboratory Fellow, Los Alamos National Laboratory
Senior Scientist, New Mexico Consortium
100 Entrada Dr, Los Alamos, NM 87544
Email: 
tterwilli...@newmexicoconsortium.org
Tel: 505-431-0010




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Re: [ccp4bb] Alexey Vagin

[Roberto Steiner]

Very very sad to hear this. I have some great memories of our time together in 
York several of which involve molecular replacement problems, Fortran77 code, 
and pubs.

A brilliant mind and a good person. Will miss having someone calling me 
'Rapierta' (Roberto pronounced with a Russian accent).

May he rest in peace in a place that he enjoys.

'Rapierta'

Sent from Android device

On 26 Mar 2023 19:29, Eugene Krissinel - STFC UKRI 
 wrote:
Dear All,

It is with a great sadness that I share with you that Alexey Vagin passed away 
this Saturday in the Radcliffe Hospital in Oxford after suffering from a heart 
attack with subsequent complications. He was 78 years old.

Alexey made many developments in methods and software for macromolecular 
crystallography, to which he devoted his whole life. He is known for his BLANC 
Suite for structure solution done at the Moscow Institute of Crystallography in 
the 1980s, as well as for his contributions to Refmac and Monomer Library. Many 
thousands of researchers have benefited from his work on Molrep and Balbes 
software for Molecular Replacement. After his retirement in 2010, Alexey 
developed and actively maintained the MorDA software for MR, which became a 
monument to his efforts.

Alexey's work will continue to serve generations of researchers through his 
contributions to CCP4, where we will take the best care of his distinguished 
legacy.

With sympathy to everyone who knew Alexey personally and for whom this is very 
sad news,

Eugene




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Re: [ccp4bb] Is this Spam? This looks like Spam.

[Roberto Steiner]

I got unsubscribed from both ccp4bb and ccpem. I also thought it was spam but 
then realised that I was not getting any message. Complained a bit with our IT 
people (which is something I tend to do anyway) then subscribed again. Since 
then received a bunch of email starting with


> SUBSCRIBE ccp4bb Roberto Steiner
You are already subscribed to the CCP4BB list as "Roberto Steiner".

> Roberto A Steiner
Unknown command - "ROBERTO". Try HELP.


…

but I am now getting ccp4bb emails.

Best wishes
Roberto


Roberto A Steiner
www.steinerlab.org<http://www.steinerlab.org>
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk<mailto:roberto.stei...@kcl.ac.uk>
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it<mailto:roberto.stei...@unipd.it>
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
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On 11 Jan 2023, at 10:15, Becker, Stefan 
mailto:s...@mpinat.mpg.de>> wrote:

You don't often get email from s...@mpinat.mpg.de<mailto:s...@mpinat.mpg.de>. 
Learn why this is important<https://aka.ms/LearnAboutSenderIdentification>
I also received this kind of message. Is it definitely spam? I waited for 24 
hours and noticed that no messages appeared under CCP4BB. Then I re-registered 
with CCP4BB. From that moment I received messages again.

Best,
Stefan Becker



Am 10.01.2023 um 15:23 schrieb Bernhard Rupp 
mailto:hofkristall...@gmail.com>>:

I got unsubscribed from ccp4em yesterday with a similar message for 
b...@ruppweb.org<mailto:b...@ruppweb.org>. That one has a minor certificate 
problem that however does not affect other email recepients.

For some reason, the ssl certificate for 
https://ruppweb.org/<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fruppweb.org%2F=05%7C01%7Croberto.steiner%40KCL.AC.UK%7C1748c303ddf14a4660ec08daf418a91d%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C638090684590397989%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=ssb42GFgpyedTeCyB6LgQYZMkrVxaE40Q5lS%2FpLe0IU%3D=0>
 is valid, but the mailserver still seems to have an issue. May be your IT can 
check yours

Best br


On Tue, Jan 10, 2023, 02:57 David Briggs 
mailto:david.bri...@crick.ac.uk>> wrote:
I received this odd-looking message from the board address. Did anyone else get 
anything similar?
[cid:b20d59ac-fc29-4cbb-b7f0-65ef44f06b97]
I'm sharing a screenshot because I'm not:
(a) going to click those links
(b) get others to click those links.

Can the board admin confirm if this is legitimate or not?

D

--
Dr David C. Briggs CSci MRSB
Principal Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fabout.me%2Fdavid_briggs=05%7C01%7Croberto.steiner%40KCL.AC.UK%7C1748c303ddf14a4660ec08daf418a91d%7C8370cf1416f34c16b83c724071654356%7C0%7C0%7C638090684590397989%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=OLeiIUCfcpk4ye0SidUbFcd%2FYhLW%2FA3sdZn0cZzlFcA%3D=0>

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Re: [ccp4bb] resolution limit stuck in Refmac5

[Roberto Steiner]

Hi Jon
On 30 Oct 2010, at 00:46, Tom Huxford wrote:


Hi all,

I'm working with good quality relatively complete x-ray diffraction
data collected to a resolution limit 2.6 Å from a crystal of a protein
with a small molecule ligand bound.  I ran MR from 10-4 Å and then did
maximum likelihood rigid body refinement in Refmac5 against data from
50-3.5 Å.  Now I would like to run restrained refinement from 50-3 Å.
The reason for doing this is that, in order to minimize the divergence
between R-cryst and R-free during refinement my advisor who, by the
way, is forwarding this e-mail for me (and editing it so please don't
bash him too mercilessly) suggested I first build in the ligand and
newly positioned polypeptide loops and refine against data at a lower
resolution limit before opening it up to all the available data.


I personally would not first build in the ligand and refine against  
low res data...

Assuming the ligand is what you're after, I would:
(a) leave the ligand out of the time being
(b) refine the model using all data
(c) build the ligand in
(d) refine the model using all data

I'm not clear why doing restrained refinement using limited data first  
should help..



Apparently this has worked well for him in the past (and it has!).   
The

problem is that I'm to the point where I'd like to extend the
resolution down to 3 Å during restrained refinement but even if I set
the range from 50-3 Å in the ccp4i window refinement only happens from
50-3.5 Å.  If I take a step back and do the rigid body refinement from
50-3 Å and then carry out restrained refinement from 50-3 Å it works
fine.  Why would the limits imposed by rigid body refinement cause the
subsequent restrained refinement to be stuck at the rigid body
refinement's resolution limits?


Are you using the original reflection file?

Best
Roberto



Thanks for any thoughts,

Jon Fleming
Graduate Student
Structural Biochemistry Laboratory (Huxford Lab)
Department of Chemistry  Biochemistry
San Diego State University


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] Deposition of riding H [was: Deposition of riding H]

[Roberto Steiner]

Dear Pavel,

Stressing Ethan's point about TLS refinement becoming practical with  
Refmac implementation, Winn et al. (2001) Acta D, 57, 122-133 (I know  
you like references) states:


Derivatives of the residual with respect to the TLS parameters are  
expanded in terms of the derivatives with respect to individual  
anisotropic U values, which in turn are calculated using a fast  
Fourier transform technique. TLS refinement is therefore fast and can  
be used routinely.


Best wishes
Roberto


On 15 Sep 2010, at 19:34, Pavel Afonine wrote:


Dear George,

a small correction if I may:


However
you will not find TLS in the index, because the credit for
implementing this very useful concept should be given to
Martin Winn, Garib and Ethan, long after the current version
of SHELXL (and its manual) were released in 1997.


Acta Cryst. (1985). A41, 426-433
Restrained structure-factor least-squares refinement of protein  
structures using a vector processing computer

I. Haneef, D. S. Moss, M. J. Stanford and N. Borkakoti

Abstract: A least-squares refinement program RESTRAIN has been  
developed, which is capable of refining macromolecular structures  
using structure amplitudes, phases from isomorphousreplacement  
or anomalous scattering and pseudo-energy restraints. In addition to  
positional parameters and isotropic temperature factors, anisotropic  
mean-square displacements may be refined either as individual atomic  
U tensors or as TLS tensors applied to groups of atoms. Anharmonic  
effects may be handled by coupling together occupancies to enable  
the electron density of an atomic group to be distributed over more  
than one subsite. A novel way of restraining groups of atoms to be  
planar has been developed that does not require dummy atoms and does  
not restrain the plane to lie in its current orientation.


One can find other, earlier programs, but they are small molecule  
specific.


Regards,
Pavel.


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] turn granular to crystal

[Roberto Steiner]

Dear Rui,

I can only support Patrick's view on MMS. We've got some very good  
results with this technique in one tricky case in which the starting  
point wasn't much better than yours.
In addition to the papers Patrick suggested the following might also  
be worth a look:


The role of bias in crystallization conditions in automated  
microseeding.

St John FJ, Feng B, Pozharski E.
Acta Crystallogr D Biol Crystallogr. 2008 Dec;64(Pt 12):1222-7.

Cheers
Roberto

On 25 Aug 2010, at 12:37, rui wrote:


Hi, All,

I'm trying to crystallize a soluble protein and got something like  
granular, they are rounded shaped and not so regular and also don't  
have sharp edges. See the attached pic. The current condition is  
PEG4000 and pH around 5. How can I improve this condition? Thanks a  
lot.



granular.JPG


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] TLSMD Alignments

[Roberto Steiner]

Dear Micheal,

As you say, the effect is probably arising from the different packing  
environment of your NCS-related molecules.
Once you've taken TLS groups into account NCS restraints might even  
perform better.


Cheers,
Roberto

On 24 Aug 2010, at 21:15, Michael Thompson wrote:


Hi All,

I have a question for those of you familiar with the TLSMD  
webserver. I am working on a structure with slightly imperfect 3- 
fold rotational NCS. My most recent .pdb file has been generated  
using Refmac (followed by a little tinkering in Coot), and during  
refinement I have been imposing medium main chain and loose side  
chain NCS restraints, and my R-factors don't really improve if I  
loosen the restraints further. This is the .pdb file I've also used  
an input to TLSMD.


The results of TLSMD do show that the residuals appear to slowly  
plateau when breaking the chains into 19 or 20 groups (all three  
A,B,C seem to converge similarly). When I look at the alignments,  
the TLS groups created for each chain do not align with each other  
well. The alignment of groups gets slightly better as more groups  
are added, which is partially just an issue of the groups being  
smaller and looking closer I think, but there is still significant  
stagger between neighboring groups. Is this typical for a structure  
with NCS-related chains? It seems somewhat counterintuitive to my  
understanding of how symmetric proteins should work (if the TLS  
motions reflect actual motions of the molecule). Perhaps the  
difference in TLS grouping between chains results from differences  
in Biso for NCS-related atoms that result from crystal packing?  
Maybe someone can shed some light on the situation?


Thanks a lot,

Mike Thompson




--
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

[ccp4bb]

[Roberto Steiner]

Dear Changyi,

Yes it can. You should have a xxx.cif file which Refmac uses to know  
about your ligand. Please have a look at it and check that it is  
actually representing the ligand you want to refine. You can
then play with standard deviations in the file to put more weight on  
idealised geometry (assuming it is correct!) in case ligand distortion  
is caused by limited resolution. In general, if ligand description is  
correct playing with SD should not be required. Clearly, make sure  
that distortion does not have a functional meaning.


Best
Roberto

On 4 Aug 2010, at 20:15, Changyi Xue wrote:


Dear all,

  In my structure, there is a ligand, which contains a sugar  
ring. In the process of refinement, refmac always tried to distort  
the sugar ring to fit into the density. Is there any way to fix or  
restrain the ring conformation more tightly? I know CNS has such  
function, just wandering if refmac could do it also.


  all suggestions are welcome.


changyi


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] Deposition of a BUSTER refined structure

[Roberto Steiner]

Dear Melanie

On 2 Mar 2010, at 15:09, Melanie Vollmar wrote:


Hi all

I did my first deposition of a BUSTER refined structure and  I was  
very surprised when I was asked for an estimated error of free R  
value and estimated error of bin free R value as well an ESD  
based on Luzzati Plot during the deposition process.


I had a detailed look into the refinement log file and I tried the  
Buster documentation but I have absolutely no clue where I can find  
these values.


Can someone give me a hint where I can find some information about  
this or where the values are in the log file? If they are not listed  
anywhere, in which way do they have to be estimated? And what is a  
Luzzati Plot? I have never heard of such a plot before...





For example, page 305-307 of Jan Drenth 'Principles of Protein X-ray  
Crystallography' Second Edition, Springer.
However, as pointed out by Cruickshank* (1999) a Luzzati (plot of R  
versus 2sin theta/lambda) was not really developed to give final errors.


*Acta Crystallogr D Biol Crystallogr. 1999 Mar;55(Pt 3):583-601.
Remarks about protein structure precision.

Best wishes
Roberto




Thank you very much.

Cheers

Melanie

Re: [ccp4bb] Low Bfactor values for waters after TLS

[Roberto Steiner]

Hi Alasdair
On 11 Sep 2009, at 09:46, Alasdair K. Mackenzie wrote:


Greetings CCP4-ers,

I have been running some TLS refinement which does wonders for my R/ 
Rfree
values, but seems to give some spurious Bfactors/ADPs for the  
waters, e.g.

B= 2.

These low Bfactors appear relatively consistent with the residual  
protein
B-factors (i.e. the pdb output from REFMAC), but when i use TLS  
analyse to
apply the TLS contribution to the total B-factors for the model, the  
low
water B values seem crazy...i apparently have a water with a B- 
factor

of 2 interacting with a side chain with a B-factor of 60

Has anyone encountered this type of thing before?


Yes.
I have reported this to Garib some time ago and I believe the active  
Refmac people are working on this.




and does anyone have a
good solution for getting around the problem?


Mhh...
Latest Refmac versions seem to consider waters directly bound to the  
protein as members of the various TLS groups.

You can override this by using
TLSD WATERS EXCLUDE

You can give it a go and see if it helps.

Best
Roberto




cheers,
Al

Re: [ccp4bb] Alternative conformation in Mac Refmac5

[Roberto Steiner]

Hi Heidi,

I have noticed the same thing. Went back to Refmac v93 (mac version)  
and side-chains stopped flying. Needs a fix.


Best
Roberto
On 11 Sep 2009, at 10:59, Heidi Tuominen wrote:


Hi all,

I have updated lately to ccp4-6.1.2. I have Macintosh. Now I have  
problem with alternative conformations. After Refmac5 run all  
alternative conformation atoms are fully unordered without any  
chemical restraints. If I put same pdb to Windows Refmac5.5.0102  
there is no any problems except R-factors are some higher.


Do I have some problem or is there bug in Mac version of  
Refmac5.5.0102? How problem could be solved?


Thank you,
Heidi Tuominen
University of Turku, Finland

Re: [ccp4bb] cyseteine modification

[Roberto Steiner]

You can also have a look at

Active site structural features for chemically modified forms of  
rhodanese.

Gliubich F, Gazerro M, Zanotti G, Delbono S, Bombieri G, Berni R.
J Biol Chem. 1996 Aug 30;271(35):21054-61.



Best
Roberto

On 19 Aug 2009, at 15:41, Debajyoti Dutta wrote:


Dear Sir,

Is there any other oxidation states of cysteine other than cysteine  
sulphinic acid and cysteine sulphonic acid. In my protein, the  
cysteine molecule is definitely overoxidized but the electron  
density is not corresponding to the sulphinic or the sulphonic acid.  
The positive density looks as if it can accomodate only one oxygen  
atom and not more.


Thank you for reply in advance.

Sincerely
Debajyoti Dutta

Re: [ccp4bb] quaternary structure comparison

[Roberto Steiner]

Hi Eike,

PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) or SCOPPI (http://www.scoppi.org 
) might be useful.

Interested in hearing about other alternatives.

Best wishes
Roberto

On 15 Jun 2009, at 21:28, Eike - Christian Schulz wrote:


Dear All,

I solved a structure that has a very common tertiary structure motif  
but it appears as if the arrangement of the monomers is somewhat  
‘non-standart’.


Of course I performed a DALI search, which resulted in a great  
number of hits  due to the similarity of the tertiary structure  
(Z’s: 250 8; 100  10).


In order to  check whether this arrangement that I see is something  
new I would like to perform a quaternary structure comparison. Is  
there any such tool available? A Google search wasn't very  
conclusive. Or is there a clever combination of available resources?


Kind regards

Eike


---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] issues with TLS refinement

[Roberto Steiner]

I might be wrong but it seems Garib has implemented some sort of line  
minimization for the tls part which causes the observed behavior. Garib?


Roberto

On 21 May 2009, at 00:39, Dima Klenchin klenc...@facstaff.wisc.edu  
wrote:



The issue is:

While running tls and restrained refinement, the program doesn't  
perform
the given no. of tls refinements. For e.g., if you ask it to  
perform 10
TLS and 5 restrained refinement rounds, it'll run may be 2 or 3  
rounds of
TLS and then jump to restrained refinement rounds and finish the  
job. It

is, however, finishing the given no. of restrained refinements
every-time.  There is no error message or any indication of abrupt  
ending

to tls refinements in the log file either.


FWIIW, I am getting the same thing. Sometimes no TLS refinement is  
done at

all, sometimes the program does 2-3 cycles and sometimes the requested
number of cycles. Seems to be PDB input-dependent but I could never  
find

what causes the behavior.

Dima

[ccp4bb] postdoctoral position at King's College London

[Roberto Steiner]

Dear ccp4bb readers,

---
King's College London
Cardiovascular Division and Randall Division of Cell and Molecular  
Biophysics


A postdoctoral position is immediately available for a highly  
motivated molecular biologist/biochemist with an interest in  
structural biology (macromolecular crystallography and NMR)
to perform structure-function studies of proteins and protein  
complexes involved in control of reactive oxygen species (ROS) in  
redox signalling.


The position is supported by a British Heart Foundation Centre of  
Excellence scheme and is part of a research collaboration between the  
groups of Drs Sasi Conte and Roberto Steiner (Randall Division of  
Cell and Molecular Biophysics) and Professor Ajay Shah  
(Cardiovascular Division). Excellent infrastructure support is  
available to carry out the project.


The salary is Grade 6, SP31 which translates into: £30,594 plus  
£3,323 (London weighting). The position is initially funded for 18  
months.


For informal enquiries please contact: roberto.stei...@kcl.ac.uk



http://www.kcl.ac.uk/schools/medicine/research/cardio/pi/shah-a.html
http://www.kcl.ac.uk/schools/biohealth/research/randall/
---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

[ccp4bb] postdoctoral position at King's College London (2)

[Roberto Steiner]

Dear ccp4bb readers,

£1,000 worth correction on my previous postdoc ad: London weighting  
is £2,323 and not £3,323.


Apologies (all the rest is correct..),
Roberto



On 9 Mar 2009, at 09:49, Roberto Steiner wrote:


Dear ccp4bb readers,

---
King's College London
Cardiovascular Division and Randall Division of Cell and Molecular  
Biophysics


A postdoctoral position is immediately available for a highly  
motivated molecular biologist/biochemist with an interest in  
structural biology (macromolecular crystallography and NMR)
to perform structure-function studies of proteins and protein  
complexes involved in control of reactive oxygen species (ROS) in  
redox signalling.


The position is supported by a British Heart Foundation Centre of  
Excellence scheme and is part of a research collaboration between  
the groups of Drs Sasi Conte and Roberto Steiner (Randall Division  
of Cell and Molecular Biophysics) and Professor Ajay Shah  
(Cardiovascular Division). Excellent infrastructure support is  
available to carry out the project.


The salary is Grade 6, SP31 which translates into: £30,594 plus  
£3,323 (London weighting). The position is initially funded for 18  
months.


For informal enquiries please contact: roberto.stei...@kcl.ac.uk



http://www.kcl.ac.uk/schools/medicine/research/cardio/pi/shah-a.html
http://www.kcl.ac.uk/schools/biohealth/research/randall/
---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk






---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] Twin refinement - selecting FreeR

[Roberto Steiner]

Hi Sabine,

If your question is: Is it possible to refine twinned structures with  
Refmac?

My answer is: Yes. Very well.

Best wishes,
Roberto

On 20 Feb 2009, at 16:32, Sabine Schneider wrote:


Hi everyone,

I got good data to 2.4A on a protein-ligand complex. I want to  
solve the

structure by MR using a model with 45% seq. ID and 55% similarity.
Initially the data appeared to be P622 (pointless, selfrotation  
function

etc). Unit cell: 145 145 65, MW protein is 28kDa
I prepared the MR model with chainsaw, choping of non-conserved  
residues

at the C gamma and reset the B-factors. Phaser found a solution, but
with negativ LLG.

I than processed the data in P3, P321, P312 and P6 and  run Phaser
searching all possible alternative spacegroups. The best solution  
is P3
(4mol/asu) followed by P321 (2mol/asu),both with a LLG of above  
400. But

when trying to refine the models in Refmac the R/Rfree stays at ~48%.

Looking at the truncate output and phenix xtriage, twinning is  
suggested

with the merohedral twin law -h, -k, l and a twin fraction of 40.3%
Using twin refine option in Refmac (5.5.0070), the R/Rfree for the  
P321
solution drops to around 33%, but the difference between R/Rfree is  
only

1%. For the solution found in P3 the R/Rfree stays at around 47%.

So I assumed that P321 is the better solution. Is the difference in
R/Rfree only 1%, because the free and work reflections are related
through the twin law?

I used phenix  to assign the free reflections putting in the twin
operators. Doing simulated annealing in phenix, I get a rather large
difference in R/Rfree of  ~7%. Well, I guess I need to do some  
tweaking

of the parameters in phenix (running the latest phenix and cci_apps).
When I than use these free reflections assigned by phenix in Refmac I
still get only 1.5% difference between R and Rfree?

So is it doable in Refmac, or is my best bet phenix? Any advice  
what is

the best way to proceed is much appreciated!

Sabine


--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry and Pharmacy
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77846
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/


---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] Off topic: Crystal degredation with age

[Roberto Steiner]

Hi Ed,

A bit late on the subject 

I have collected atomic resolution data (around 0.97A) on both bovine  
and porcine phospholipase A2 crystals which at
the time of data collection were between 10-16 years old.  
Crystallization setup was liquid-liquid diffusion in glass capillaries.


Differently from the other stories, here there's no degradation  
involved...just rock-solid Xtals.
In the paper we stated: 'Crystals are stable in the crystallization  
solution...' which I guess well reflects their behavior.


Best wishes,
Roberto

On 5 Feb 2009, at 19:11, Edward Snell wrote:


/lurk_mode_off
/dumb_question_on

Dear All,

I was recently trying to find references on how age may degrade a
crystal, i.e. grow them and use them or preserve them as fresh as
possible. I seem to remember seeing a couple of papers on this but my
memory is fading and I have been unable to locate them. Can anyone jog
my memory or tell me if I'm imagining things?  I've found plenty on  
the

protein prep etc. but nothing on the crystal.

Thanks,

Eddie.


Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Email: esn...@hwi.buffalo.edu  Telepathy: 42.2 GHz

Heisenberg was probably here!Crystallization, how quaint!

/dumb_question_off
/lurk_mode_on


---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk

Re: [ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?

[Roberto Steiner]

Hi Francis,
I would consider the possibility of a orthorhombic sg (a~b with  
twinning).  If unlucky maybe even monoclinic.


Cheers
Roberto





On 5 Feb 2009, at 18:10, Francis E Reyes francis.re...@colorado.edu  
wrote:



It seems like this space group will be the death of me.

I'm working on a structure in SG P41212 one molecule per asu that was
solved with experimental SAD phases.  The resolution is to 2.5 and the
refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of
0.011 and angles of 1.597 . The unit cell is 73.604   73.604  114.279
90.00  90.00  90.00.

I'm considering the case of perfect twinning where the real s.g. is
P41 masked under the higher symmetry in P41212.

It seems to be the case in perfect twinning that the approach is to
molecular replace the refined model into the lower space group. I
reindexed my data to the lower space group P41 and molecular replaced
into the reindexed data with Phaser. A single was solution was found
with 2 mol per asu (39.6% solvent content) related by NCS.
I've refined the now two fold ncs related structure in P41 to a much
more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and
angles at 0.865 refining with a twin law and NCS as implemented by
phenix.refine.

However I'm not happy:
[1] a simmulated anneal omit map one of the monomers in P41 where 5
residues in a non crystal contact region of the molecule (I wanted to
challenge the omit map) shows nearly no density. (the SA OMIT map was
generated with phenix.autobuild using the same refine parameters as
the final round of refinement)
[2] the NCS selection is a little bit troubling. (maybe the phenix
developers can chime in on this)
  reference = chain 'B' and (resseq 243:293 or resseq 310:370 )
  selection = chain 'A' and (resseq 243:293 or resseq 310:370 )
seems as if resseq 243:293 is behaving differently than 310:370?
[3] the densities of the side chains of a helix (not an xtal contact)
are poorly defined, with geometry for the backbone not so good.


There's talk about choosing the correct MR solution (see On the
molecular-replacement problem in the presence of merohedral twinning:
structure of the N-terminal half-molecule of human lactoferrinW. A.
Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use
phaser to pick my MR solution for P41. Could phaser possibly have
chosen poorly?

Thanks!

FR


-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] generating omit maps

[Roberto Steiner]

There used to be a program called OMIT in CCP4. Seems to be still  
supported.
I believe I used it years ago but I vaguely remember problems with  
some space groups (might be wrong though...)


Regards,
Roberto

On 11 Dec 2008, at 16:20, rajan sreekanth wrote:



Hi
What about the SFcheck omit map calculation in 'Map and Mask  
utilities' module in CCP4?



R.Sreekanth

On Wed, 10 Dec 2008 Kathleen Frey wrote :
Hi Everyone,

Can anyone tell me a relatively easy way to generate an omit  
density map for
a ligand? I know that CNS can do this, but I was wondering if  
there's a CCP4

related program to generate omit maps.

Thanks,
Kathleen




---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

[ccp4bb] Coot and Hs

[Roberto Steiner]

Dear all,

a problem possibly at the coot/mmdb interface...

If one uploads a pdb file (from phenix.refine in the example below)  
that contains Hs into Coot and then writes it out (with or without  
any modification done on it)  Coot shifts the HG2n of THR on the  
right by one column space. Because column 17 is kept empty

the result is three identical HG2 THR protons.

Example:
Uploaded:
CRYST1   45.460  166.970  167.200  90.00  90.00  90.00 P 21 21 21
SCALE1  0.021997 -0.00 -0.000.0
SCALE2  0.00  0.005989  0.000.0
SCALE3  0.00  0.00  0.0059810.0
ATOM  1  N   THR A   2  27.439  61.155  24.800  1.00  
70.37  AN
ANISOU1  N   THR A   2 9721   7539   9478   1186   2103 
297  AN
ATOM  2  CA  THR A   2  26.489  62.215  24.456  1.00  
77.21  AC
ANISOU2  CA  THR A   210790   8621   9925   1145   1971 
285  AC
ATOM  3  CB  THR A   2  26.960  63.618  24.975  1.00  
73.93  AC
ANISOU3  CB  THR A   210519   8251   9322   1244   1638 
409  AC
ATOM  4  OG1 THR A   2  28.259  63.512  25.579  1.00  
73.16  AO
ANISOU4  OG1 THR A   210309   7971   9516   1332   1549 
489  AO
ATOM  5  CG2 THR A   2  25.969  64.197  25.999  1.00  
64.65  AC
ANISOU5  CG2 THR A   2 9547   7174   7845   1451   1403 
582  AC
ATOM  6  C   THR A   2  26.200  62.288  22.934  1.00  
71.92  AC
ANISOU6  C   THR A   210101   8073   9151869   2179  
63  AC
ATOM  7  O   THR A   2  25.172  61.797  22.455  1.00  
64.74  AO
ANISOU7  O   THR A   2 9210   7258   8131790   2339 
-23  AO
ATOM  8  HA  THR A   2  25.639  62.015  24.900  1.00  
71.84  AH
ATOM  9  HB  THR A   2  27.009  64.232  24.226  1.00  
69.26  AH
ATOM 10  HG1 THR A   2  28.232  62.991  26.212  1.00  
67.50  AH
ATOM 11 HG21 THR A   2  26.275  65.055  26.303  1.00  
61.19  AH
ATOM 12 HG22 THR A   2  25.104  64.300  25.596  1.00  
61.19  AH
ATOM 13 HG23 THR A   2  25.894  63.607  26.753  1.00  
61.19  AH



Written:
CRYST1   45.460  166.970  167.200  90.00  90.00  90.00 P 21 21 21
SCALE1  0.021997 -0.00 -0.000.0
SCALE2  0.00  0.005989  0.000.0
SCALE3  0.00  0.00  0.0059810.0
ATOM  1  N   THR A   2  27.439  61.155  24.800  1.00  
70.37  AN
ANISOU1  N   THR A   2 9721   7539   9478   1186   2103 
297  AN
ATOM  2  CA  THR A   2  26.489  62.215  24.456  1.00  
77.21  AC
ANISOU2  CA  THR A   210790   8621   9925   1145   1971 
285  AC
ATOM  3  CB  THR A   2  26.960  63.618  24.975  1.00  
73.93  AC
ANISOU3  CB  THR A   210519   8251   9322   1244   1638 
409  AC
ATOM  4  OG1 THR A   2  28.259  63.512  25.579  1.00  
73.16  AO
ANISOU4  OG1 THR A   210309   7971   9516   1332   1549 
489  AO
ATOM  5  CG2 THR A   2  25.969  64.197  25.999  1.00  
64.65  AC
ANISOU5  CG2 THR A   2 9547   7174   7845   1451   1403 
582  AC
ATOM  6  C   THR A   2  26.200  62.288  22.934  1.00  
71.92  AC
ANISOU6  C   THR A   210101   8073   9151869   2179  
63  AC
ATOM  7  O   THR A   2  25.172  61.797  22.455  1.00  
64.74  AO
ANISOU7  O   THR A   2 9210   7258   8131790   2339 
-23  AO
ATOM  8  HA  THR A   2  25.639  62.015  24.900  1.00  
71.84  AH
ATOM  9  HB  THR A   2  27.009  64.232  24.226  1.00  
69.26  AH
ATOM 10  HG1 THR A   2  28.232  62.991  26.212  1.00  
67.50  AH
ATOM 11  HG2 THR A   2  26.275  65.055  26.303  1.00  
61.19  AH
ATOM 12  HG2 THR A   2  25.104  64.300  25.596  1.00  
61.19  AH
ATOM 13  HG2 THR A   2  25.894  63.607  26.753  1.00  
61.19  AH


Does anyone know (Paul?) the reason for the above?

The only solution right now (that I know of) is to remove all Hs and  
generate them again (molprobity for example) prior to refinement.


Cheers,
Roberto

---
Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] Setting drops with proteins that are hard to concentrate

[Roberto Steiner]

I have a similar problem Matthew.
Just got some NVoy from Novexin in which some claim can help if  
hydrophobic patches are the main problem. Will see.


Regards,
Roberto

On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote:


Dear All,

I have a 50kDa protein that is soluble and monodisperse at up to  
approx 1mg/ml (after Ni-affinity and size-exclusion chromatography).


However, it aggregates (probably both via disulphides and via  
'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ 
ml, even in the presence of several detergents. I don't want to add  
DTT since my protein should have several intramolecular  
disulphidesalthough I do have 2 free Cysteines, partially  
exposed. I have already tried mutating the Cysteines, with little  
improvement.


Any suggestions for obtaining 5-10mg/ml?

Does anybody have good experiences with usin L-Arg and L-Glu (e.g.  
At 50mM)  to aid concentrating (as in the Golovanov AP paper, JACS,  
2004, pages 8933...)


Thanks for any input!

Yours,

Matt


Matthew J. Bottomley, Ph.D.
Senior Research Biochemist
IRBM / MRL-Rome


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---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] A babyish question on coot

[Roberto Steiner]

Hi Yanming,

I am glad that at least one other person has encountered this problem 
so I do not feel too guilty for having asked Paul to do something about 
it. Anyway, the MAN was kind enough to add a masking function under the
Extensions Menu. It is something called 'Mask map bla bla..'. (I am not 
in the office so I can't read the proper name of the function). It uses 
mmdb code to define Chains/atoms/ that you want to exclude from map 
calculations and produces a map masked by them. (For example if all 
your metal centres are chain M something like M/* removes all of them). 
Very useful imo for refining metal centers at low resolution.


Roberto




Quoting Yanming Zhang [EMAIL PROTECTED]:


Hi,

After JED's email I tried immediately:
case:
a metal site which has very strong density and  a HIS co-ordinating 
with the ion. After I click 'Real space refine zone' and then  the 
residue HIS, the HIS will move to the density which belongs to the 
ion(clash with the ion) no matter how low the weight was set.


PS, I remember that Xfit of XtalView has the beauty to avoid this 
kind of problem, but now I am a regular user of coot.


Thanks
Yanming


On Tue, 29 May 2007, Debreczeni, Judit wrote:

Unreasonable geometry can be avoided by setting the refinement 
weight to a lower value -- either using Extensions-Set Matrix 
(Refinement Weight) or in your .coot file:

(set-matrix value)

There's also a coot-bb, btw.

JED






-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
Yanming Zhang
Sent: 29 May 2007 01:50
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] A babyish question on coot


Hi,

I use coot almost around the clock. One thing troubles me is that:
when clicking on 'real space refine zone', coot seems only care about the
'real space'(Electron density), sometimes it will bring the model to
the density no matter whether the density was already  claimed by other
model atoms or not, resulting in clash and unreasonable geometry. How can
I avoid this?

Sorry for an old crystallographer to ask so babyish question. But your
help can save me lots of time.
Thanks
Yanming







--
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] Stop Refmac from refining B factors?

[Roberto Steiner]

On 18 Apr 2007, at 14:39, Eva Kirchner wrote:







(But I'm still curious about the B-factor refinement when there is  
no REFI BREF ISOT in the com-file...)


Eva,

Refmac internal default is REFI BREF ISOT
that's why even if you remove the above line from the com file (or  
deselect that option from the interface) it still does ISOT BREF  
refinement.

It does tell you that though
if you look at the log file there's a bit that says

...
Method of minimisation : Sparse Matrix
  Experimental sigmas used for weighting
  Number of Bins and width:20   0.0080
  Refinement of individual isotropic Bfactors
..
what do you have there when you deselect the B fact option from the  
interface?



I agree with Herman that at 3.2 A isotropic B values refinement can  
be useful.



Roberto



Eva



2007/4/18, Mischa Machius [EMAIL PROTECTED]:
Like Harry said, it is not justified to do individual B factor  
refinement at that resolution. Well, you can do it, but you'll end  
up with funny results, such as what are observing right now. Still,  
from a pragmatic point of view, individual B factor refinement in  
cases like these can have a positive effect on the electron  
density. However, keep in mind that the resulting B factors may  
physically not be very meaningful. In the end, you'll have to  
switch to grouped B factor refinement, or you risk nasty comments  
from an attentive mentor or reviewer (and rightly so). Hope that  
helps. Best - MM


On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote:


Hi,

I have a little problem with B-factor refinement. I'm using the  
CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I  
tried 8-3.2 A as well, it doesn't make a big difference for this  
problem), and a current Rfree of 30.4%.


Refmac refines the B-factors so that they are nearly the same for  
main chain and side chain, and I don't like that (or could it make  
sense in any way?). Moreover, my structure is a protein complex,  
and Refmac is mainly doing this for one component of the complex.  
If I take the B-factors from the original uncomplexed protein  
(around 18, 1.75 A) and add 44 to them with moleman to get them in  
the range they are in the complex, Refmac flattens them  
remarkably in only 5 cycles of restricted refinement. Does anyone  
have an explanation for this? I am pretty sure that the complex  
components are in the right place, I see beautiful density and  
everything I should see at this resolution.


Here is what I tried further:

* I de-selected Refine isotropic temperature factors in the  
Refmac interface. There was no REFI BREF ISOT any more in the com  
file. But there was also no difference in the B-factors compared  
to when there _was_ REFI BREF ISOT in the com file... So does  
Refmac just _ignore_ my wish not to refine B-factors? (The REFI  
keywords were as follows: type REST - resi MLKF - meth CGMAT - is  
there any B-factor-thing hidden in this?)


* I played around with the geometric parameters. If I select the B- 
factor values there (the keywords are TEMP|BFAC  
wbskalsigb1sigb2sigb3sigb4), it does not make _any_  
difference, what values I fill in there, the resulting B-factors  
are always the same (but different from when I don't use the TEMP  
keyword, and even flatter). Default for WBSCAL is 1.0, I tried  
10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs.


Thanks for any thoughts on this,

Eva



-- 
--

Mischa Machius, PhD
Associate Professor
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353





---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] Question about glove boxes for protein crystallization

[Roberto Steiner]

Hi Mathews,

An anaerobic chamber from Belle Technology (http://www.belle- 
technology.com/) served me well in the past.
Belle's glove boxes are made of acrylic material. That offers  
relatively low-cost and all-round visibility.


Cheers,
Roberto

On 23 Feb 2007, at 02:03, Mathews, Irimpan wrote:


Dear Friends,

Sorry for the non CCP4 question. We are planning to purchase a  
small glove box to setup crystallization trays under anaerobic  
conditions. If you have used glove boxes for crystallization, would  
you please give me some idea?


We are thinking of getting the 815 series from Plas-labs (link below).

http://www.plas-labs.com/

Thank you very much,
Mathews

Ps: If others are interested, I will post a summary.


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] how many stuck datasets are actually twinned?

[Roberto Steiner]

Hi Sue,

This reference has very useful info

Lebedev AA, Vagin AA, Murshudov GN.

Intensity statistics in twinned crystals with examples from the PDB.
Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):83-95. Epub  
2005 Dec 14.

PMID: 16369097 [PubMed - indexed for MEDLINE]


Roberto


On 21 Feb 2007, at 15:22, Sue Roberts wrote:


Hello

A partially philosophical, partially pragmatic question.

I've noticed a trend, both on ccp4bb and locally, to jump to  
twinning as an explanation for data sets which do not refine well -  
that is data sets with R and Rfree stuck above whatever the  
person's pre-conceived idea of an acceptable R and Rfree are.
This usually leads to a mad chase through all possible space  
groups, twinning refinements, etc. and, in my experience, often  
results in a lot of time being spent for no significant improvements.


Just out of curiosity, does  anyone have a feel for what fraction  
of stuck data sets are actually twinned? (I presume this will vary  
somewhat with the type of problem being worked on).


And a  sorta-hypothetical question, given nice-looking crystals;  
images with no visible split spots, extra reflections, or streaks;  
good predictions; nice integration profiles; good scaling with  
reasonable systematic absences; a normal solvent content; and a  
plausible structure solution,  and R/Rf somewhat highish (lets say . 
25/.3  for 1.8 A data), how often would you expect the Stuck R/Rf  
to be caused by twinning (or would you not consider this a failed  
refinement).  (My bias is that such data sets are almost never  
twinned and one should look elsewhere for the problem, but perhaps  
others know better.)


Sue
Sue Roberts
Biochemistry  Biopphysics
University of Arizona

[EMAIL PROTECTED]


---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]