Re: [ccp4bb] How to make an isopeptide bond?

[Isupov, Michail]

Hi Ronnie,
you will need to create a dictionary of the link in Jligand or Acedrg (ccp4i2)
if we talk about ccp4 programs, and provide this as a LIBIN input in Refmac5.


In coot you will need to delete ND2 atom of Asn residue (if your dictionary
contains link Lys NZ - Asn CG) and in menu calculate/modellling/make link
join NZ and CG atoms.

Best wishes,
Misha Isupov

From: CCP4 bulletin board  on behalf of Ronnie Berntsson 

Sent: 16 March 2022 14:57
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] How to make an isopeptide bond?

Dear all,

I have a protein structure that has 4 isopeptide bonds (between Lys and Asn 
pairs). However, I can’t for the life of me figure out a way to model this in 
in Coot (or by editing the pdb file, or some other method) so that I can refine 
it properly. Googling it hasn’t really helped much either, so I was hoping that 
someone here might know of an (preferably easy) way to do this?

Best wishes,
Ronnie

--
Ronnie Berntsson, PhD
Member of the Young Academy of Sweden
Chair of the Swedish Society for Biochemistry, Biophysics and Molecular biology

Department of Medical Biochemistry and Biophysics, Umeå University
90187 Umeå, Sweden
e-mail: ronnie.bernts...@umu.se
phone: +46 90 7865235
web: https://www.biostruct.umu.se/principal-investigators/ronnie-berntsson/




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[ccp4bb] Shortest distance between side chains

[Isupov, Michail]

Hi

I am wondering if there  is a simple program that would convert a pdb file into 
a matrix that

shows the smallest distance between all pairs of amino-acid side chains?

 Thanks,
Misha



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Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)

[Isupov, Michail]

Hi Guto,

I would be very cautious with the InnerShell merging statistics as yours.
Rmeas in this shell is higher than Rmeas overall,
which probably means you have a lot of overloads,
(or problems with backstop shadow, which is not so likely
on Diamond nowdays).

 And generally InnerShell Rmerge over 5-6% in XDS processing
 (Diamond pipelines) is usually an indicator of future problems in 
refinement.

Rfree of 25 % at 1.06  A seems to confirm your space group. 
In the case of  wrong space group or origin (ZANUDA), it would normally be well 
over 35 %.

Try to collect data  setting transmission/exposure time to lower values.
 If this was the only crystal ever explain high-ish value of freeR in the 
methods
session as due to overexposure.

For cases like yours I would really like to see Rmeas in the inner shell in 
Table 1.

Best wishes,

Misha Isupov


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Guto Rhys 
[guto.r...@bristol.ac.uk]
Sent: Tuesday, October 9, 2018 6:12 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset 
(1.05 Ang)

Hi all,

I have a 1.05 Angstrom dataset that I was able to phase but the refined model 
only has an Rfree of approximately 0.25. The dataset includes 1800 images and, 
as the crystal did not suffer significantly from radiation damage, comprises 
all 360 deg. Auto-processing pipelines at diamond light source all suggest 
I222. I have also indexed the data in iMOSFLM, which has the highest-symmetry 
Laue group that is least penalised of I422. Subsequent scaling and merging in 
AIMLESS strongly indicates that I222 is the likely space group (see below). I 
have ran the refined model through ZANUDA, which has similar R values to lower 
symmetry space groups (see below). The output from Phenix Xtriage does not find 
any specific crystal pathologies and if twinning is present it is very low (2 
to 4%, see below). The difference map suggests that the model accounts for 
nearly all the density. Any ideas or direction would be greatly appreciated.

Best,
Guto


AIMLESS Summary
   Overall  InnerShell  OuterShell
Low resolution limit   27.75 27.75  1.07
High resolution limit   1.05  5.75  1.05

Rmerge  (within I+/I-) 0.050 0.078 0.466
Rmerge  (all I+ and I-)0.051 0.080 0.536
Rmeas (within I+/I-)   0.055 0.086 0.591
Rmeas (all I+ & I-)0.054 0.085 0.613
Rpim (within I+/I-)0.023 0.034 0.359
Rpim (all I+ & I-) 0.017 0.028 0.288
Rmerge in top intensity bin0.049- -
Total number of observations  107950   779  1972
Total number unique1131588   486
Mean((I)/sd(I)) 19.7  46.2   1.8
Mn(I) half-set correlation CC(1/2) 0.998 0.994 0.796
Completeness99.1  99.2  90.4
Multiplicity 9.5   8.9   4.1

Anomalous completeness  98.1 100.0  79.1
Anomalous multiplicity   5.0   6.4   2.2
DelAnom correlation between half-sets -0.067 0.286 0.097
Mid-Slope of Anom Normal Probability   0.789   - -

Estimate of maximum resolution for significant anomalous signal =  1.14A, from 
CCanom >  0.15

Estimates of resolution limits: overall
   from half-dataset correlation CC(1/2) >  0.30: limit =  1.05A  == maximum 
resolution
   from Mn(I/sd) >  1.50: limit =  1.05A  == maximum 
resolution
   from Mn(I/sd) >  2.00: limit =  1.07A

Estimates of resolution limits in reciprocal lattice directions:
  Along h axis
   from half-dataset correlation CC(1/2) >  0.30: limit =  1.06A
   from Mn(I/sd) >  1.50: limit =  1.09A
  Along k axis
   from half-dataset correlation CC(1/2) >  0.30: limit =  1.11A
   from Mn(I/sd) >  1.50: limit =  1.13A
  Along l axis
   from half-dataset correlation CC(1/2) >  0.30: limit =  1.05A  == maximum 
resolution
   from Mn(I/sd) >  1.50: limit =  1.05A

Anisotropic deltaB (i.e. range of principal components), A^2:  6.40

Average unit cell:   29.12   29.26   55.50   90.00   90.00   90.00
Space group: I 2 2 2
Average mosaicity:   0.36

AIMLESS Laue Group prediction

   Laue GroupLklhd   NetZc  Zc+   Zc-CCCC-  Rmeas   R-  Delta 
ReindexOperator

= 1I m m m  ***  0.987   6.25  9.19  2.94   0.92  0.29   0.07  0.49   0.0 
[h,k,l]
  2  I 1 2/m 1   0.004   4.36  9.32  4.96   0.93  0.50   0.07  0.30   0.0 
[-h,-k,l]
  3  I 1 2/m 1   0.004   4.14  9.24  5.10   0.92  0.51   0.07  0.30   0.0 

Re: [ccp4bb] NMR or Homology Model as a MR model

[Isupov, Michail]

Dear Nishant,

For successful MR a choice of domain boundaries and loop trimming is
crucial.
Last couple of years I do not even try to run molrep/phaser before
running MrBump and Morda, which do a good job with model preparation.
Only when these pipelines give leads, but not quite the results I use
molrep/phaser/amore.

Morda also prepares NMR-like models (assemblies) from homology models.

Regards,
Misha





From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andreas Forster 
[docandr...@gmail.com]
Sent: Tuesday, August 29, 2017 12:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] NMR or Homology Model as a MR model

Dear Nishant,

Rosetta is a good suggestion.  You can also use an ensemble of several related 
(superposed) structures as your search model.  This will improve your chances 
of success.

All best.


Andreas



On Tue, Aug 29, 2017 at 12:50 PM, Nishant Varshney 
> wrote:
Dear Crystallographers,

I am working to solve an human protein structure which has a domain sequence 
identity of 24% with domain of another protein.  As Phaser as well as Molrep 
failed to give any definite solution (TFZ=3.7 from MR), I want to ask, if 
solution structure of another protein having domain sequence similarity of 25% 
or a homology model can be used as a template for MR?
Many thanks and Regards
Nishant

--
Dr. Nishant Kumar Varshney,
Research Associate,
C/O Dr. Sameena Khan,
Drug Discovery Research Center,
Translational Health Science and Technology Institute (THSTI)
NCR Biotech Science Cluster,
3rd Milestone, Faridabad – Gurgaon Expressway,
Faridabad – 121001 (HARYANA), India
Ph: +91- 0129-2876477
Mob: 8390564690

Re: [ccp4bb] No improvement in R-factor after Refmac.

[Isupov, Michail]

Hi,

I have seen cases where in a correct space group 
'R-work and R-free values 0.25 and 0.32 respectively'
at 2 A resolution sound like not too bad values.
In some of such cases when data from a different crystal
in the same space group was available R-factors were much lower
when the structure was refined against the new crystal data.
I guess this phenomenon could be due to uneven freezing of the first crystal,
or inconsistent degree of disorder between crystals.

In other projects high R-factor values (e.g. FreeR around 33% at 2.1 A 
resolution)
 are consistent through a range of crystals
even when the refinement is in P1, although the map quality is good enough
to see cofactors and to build  the missing parts of the structure (30% of 
residues). 
The disorder seems to be an intrinsic property of such crystal form.

I do not know how to approach publishing these results since most referees will 
argue
that such R-factors may be acceptable at 4A resolution but not close to 2 
Angstrom.

Best wishes,

Misha Isupov

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Randy Read 
[rj...@cam.ac.uk]
Sent: Saturday, March 18, 2017 9:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] No improvement in R-factor after Refmac.

Hi,

I was just going to make the same point!  The only thing to add is that, if 
there really is translational NCS (which is certainly possible with 4 copies in 
the a.u.), then it’s essential both to account for it (which current versions 
of Phaser should do automatically, if you search for all 4 copies in one job) 
and to try all possible space groups.  The situation Craig describes, in which 
it’s not immediately obvious whether your crystal has a crystallographic 2(1) 
and a pseudosymmetric non-crystallographic 2-fold or the reverse, is not 
uncommon.  However, we’ve found that the likelihood score accounting for the 
effect of tNCS is pretty good at discriminating the two possibilities.

Best wishes,

Randy Read

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

> On 18 Mar 2017, at 06:12, CRAIG A BINGMAN  wrote:
>
> You really need to approach such situations with caution.  Examination of the 
> relatively small number of axial reflections probably show that there might 
> be twofold screw axes in all three directions.  But a non-crystallographic 
> microscopic translation of nearly 0.5 in the direction of a crystallographic 
> axis will give the same pattern of strong and weak reflections as a 
> crystallographic twofold screw axis.  If I were you, I would be very sure to 
> try molecular replacement in all possible orthorhombic space groups.  Several 
> programs, including Phaser, will organize that exhaustive search across all 
> eight possibilities for you.
>
>> On Mar 17, 2017, at 11:56 PM, Polisetty Satya Dev  wrote:
>>
>> Hi,
>>
>> We checked all possible space groups of orthorhombic crystal system using 
>> Scala and Pointless but the statistics show that P212121 is the possible 
>> space group.
>>
>> Thank You,
>> Satya Dev
>>
>> On Fri, Mar 17, 2017 at 8:03 PM, Teplyakov, Alexey [JRDUS] 
>>  wrote:
>> Check the space group. It may be orthorhombic with a pure rotational axis 
>> (e.g. P21212) or even monoclinic.
>>
>>
>>
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
>> Polisetty Satya Dev
>> Sent: Friday, March 17, 2017 9:51 AM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [EXTERNAL] [ccp4bb] No improvement in R-factor after Refmac.
>>
>>
>>
>> Dear all,
>>
>> I solved a structure at 2.0 A resolution with R-work and R-free values 0.25 
>> and 0.32 respectively and I am stuck at Refmac step where there is no 
>> further reduction in R-factor.
>>
>> The above stated values were obtained after several rounds of manual 
>> refinement followed by refmac. There are also areas where electron density 
>> is missing around peptide backbone in one of the monomer in ASU.
>>
>> Can anyone please tell me how can I improve the electron density and 
>> R-factor.
>>
>>
>> The structure solution was obtained using Phaser MR and here are the data 
>> statistics:
>>
>>
>>
>> Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
>> Space group: P212121,
>> Completeness 99.5,
>> Multiplicity 6.4,
>> Four monomers per ASU.
>> Solvent content: 47%.
>>
>> Thank you everyone,
>> Satya Dev,
>> JNCASR.
>>
>

Re: [ccp4bb] ccp4 release 7.0 update 023

[Isupov, Michail]

Hi,
I have already uninstalled the latest update (23), on Snow Leopard 
the new COOT was crushing
on attempt to mutate residue in graphical interface.
Regards,
Misha



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of wtempel 
[wtem...@gmail.com]
Sent: Wednesday, November 23, 2016 3:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ccp4 release 7.0 update 023

Hello,
after installing updates 22 and 23 on an Ubuntu-14.04 (x86_64) box, I have yet 
to successfully “mutate” any amino acid residue in COOT.
(setup-mutate 1) is printed to the terminal, followed by a line confirming on 
which atom I have clicked, but the residue type selection dialog does not 
appear. Coot just hangs at this point. Has anyone else encountered this problem?
Regards.
Wolfram

​

On Wed, Nov 23, 2016 at 9:41 AM, Charles Ballard 
> wrote:
Dear All

ccp4 update 23 has just been released.  It contains

  * coot:
 - update to coot 0.8.7 on linux and os x

  * phaser:
- Bug fixes: ccp4i and interaction with Arcimboldo
- Update: Improved treatment of systematically weak data (e.g. severe 
anisotropy)

  * ccp4i
 - bug fix: phaser, updated defaults for phaser tasks
 - bug fix: mrbump, updated defaults for phaser

All the best

Charles  Ballard on behalf of CCP4 core team

Re: [ccp4bb] Problem with jligand in CCP4 7.0

[Isupov, Michail]

It also works on my up-to-date mac version of CCP4 7.0.
Misha


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Robbie Joosten 
[robbie_joos...@hotmail.com]
Sent: Tuesday, July 5, 2016 5:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problem with jligand in CCP4 7.0

Have you updated CCP4 lately? For my version 64-bit Linux the problem was fixed 
by an update a while ago. Perhaps in your case, there is a mix-up in 
environment values.

HTH,
Robbie

Sent from my Windows 10 phone

Van: Anna Gardberg<mailto:anna.s.gardb...@gmail.com>
Verzonden: dinsdag 5 juli 2016 17:57
Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Onderwerp: Re: [ccp4bb] Problem with jligand in CCP4 7.0

I've also encountered this problem recently; the simplest solution that worked 
was to start a new terminal window, sourcing an older ccp4-6.4.0 installation. 
Is there any plan to update JLigand to enable it to work with 7.0?

On Wed, Feb 10, 2016 at 7:14 AM, Robbie Joosten 
<robbie_joos...@hotmail.com<mailto:robbie_joos...@hotmail.com>> wrote:
Hi Petr,

If you are desperate, you can reformat the mon_lib_list.cif file to the old 
format. I'm guessing this would sort the problem out.

Cheers,
Robbie

Sent with my Windows Phone

Van: Petr Leiman<mailto:petr.lei...@epfl.ch>
Verzonden: ‎10-‎2-‎2016 12:36
Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Onderwerp: Re: [ccp4bb] Problem with jligand in CCP4 7.0

Dear All,

I have to revive this thread because update 7.0.001 changes the monomers 
library in a way that jligand cannot read it any more. The only solution is to 
not install this update. Sorry I cannot provide additional details on which new 
“feature” in the library upsets jligand, but it would be very nice to have this 
resolved…

Sincerely,

Petr

--
Petr Leiman
EPFL
BSP 415
CH-1015 Lausanne
Switzerland
Office: +41 21 69 30 441
Mobile: +41 79 538 7647<tel:%2B41%2079%20538%207647>
Fax: +41 21 69 30 422
http://lbbs.epfl.ch

> On Feb 1, 2016, at 19:25, Pedro Matias 
> <mat...@itqb.unl.pt<mailto:mat...@itqb.unl.pt>> wrote:
>
> Thanks, there must be something wrong with the update.
>
> Às 18:13 de 01/02/2016, Isupov, Michail escreveu:
>> As a temporary fix  I have uninstalled the latest update
>> and jligand works again.
>> Cheers,
>> Misha
>>
>> 
>> From: CCP4 bulletin board 
>> [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] on behalf of matias 
>> [mat...@itqb.unl.pt<mailto:mat...@itqb.unl.pt>]
>> Sent: Friday, January 29, 2016 10:27 PM
>> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
>> Subject: [ccp4bb] Problem with jligand in CCP4 7.0
>>
>> Dear CCP4ers,
>>
>> In CCP4 7.0 (updated) I get an error message every time I try to start 
>> jligand from the command line (see attached image). Does anyone know how to 
>> fix this problem. I am forced to revert back to version 6.4 whenever I need 
>> to use jligand.
>>
>> I AM my system administrator and I have no idea of what's happening...
>>
>> Thanks in advance,
>>
>> Pedro
>>
>>
>
>
> --
>
> Industry and Medicine Applied Crystallography
> Macromolecular Crystallography Unit
> ___
> Phones : (351-21) 446-9100 Ext. 1669
> (351-21) 446-9669 (direct)
> Fax   : (351-21) 441-1277 or 443-3644
>
> email : mat...@itqb.unl.pt<mailto:mat...@itqb.unl.pt>
>
> http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography
> http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit
>
> Mailing address :
> Instituto de Tecnologia Quimica e Biologica
> Universidade Nova de Lisboa
> Av. da República - EAN
> 2780-157 Oeiras
> PORTUGAL
>
> ITQB, a great choice for your PhD
> https://youtu.be/de6j-aaTWNQ
>
> Master Programme in Biochemistry for Health
> https://youtu.be/UKstDCFjYI8
>
> BioStruct-X Workshop on Ensemble Refinement of Protein Crystal Structures
> http://www.itqb.unl.pt/er2016

Re: [ccp4bb] How many is too many free reflections?

[Isupov, Michail]

Hi Graeme,

in a data set with just below  800,000 independent reflections I use 1 % for 
freeR which
is still impressive 8,000. xia2 would have assigned 40,000 for freeR
at 5 %. I think this is way too much.

Often we collect many data sets of the same project to find the better data.
We do use default xia2 FreeR assignments at this stage, and after locating the 
best
data set we can not go back and reassign FreeR, as the new set will be biased 
towards
the model.
Referees/editors however query cases when over 5,000 reflections were used for 
cross-validation.

Misha



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pavel Afonine 
[pafon...@gmail.com]
Sent: Tuesday, June 2, 2015 3:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How many is too many free reflections?

Hi Graeme,

free reflections are used for two purposes, at least: cross-validation 
(calculation of Rfree) and ML parameters estimation (sigmaa or alpha/beta). For 
the latter it is important that each relatively thin resolution bin 
(sufficiently thin so that alpha/beta can be considered constants in it) 
receives no less than 50 reflections absolute min; in Phenix we found that ~150 
per bin is sufficient and this is what's used by default.

Pavel

On Tue, Jun 2, 2015 at 3:26 AM, Graeme Winter 
graeme.win...@gmail.commailto:graeme.win...@gmail.com wrote:
Hi Folks

Had a vague comment handed my way that xia2 assigns too many free reflections 
- I have a feeling that by default it makes a free set of 5% which was OK back 
in the day (like I/sig(I) = 2 was OK) but maybe seems excessive now.

This was particularly in the case of high resolution data where you have a lot 
of reflections, so 5% could be several thousand which would be more than you 
need to just check Rfree seems OK.

Since I really don't know what is the right # reflections to assign to a free 
set thought I would ask here - what do you think? Essentially I need to assign 
a minimum %age or minimum # - the lower of the two presumably?

Any comments welcome!

Thanks  best wishes Graeme

Re: [ccp4bb] non-specific disulfide bonds in crystal structures

[Isupov, Michail]

Hi Todd,

disulfides (both intra and intermolecular) are quite common in extremophile
cytosolic proteins. I routinely come across these.
Many of these disulfides are retained when the proteins are overexpressed in 
E.coli regardless of the
strong reducing environment.
Usually these disulfides are relatively buried and the attack by  reducing 
molecules
such as thioredoxin is hindered.
Many of them are only partially reduced when mercaptoethanol or are reducing 
agents are used in purification
and the structures have to be modelled with partial occupancies of 
reduced/oxidised disulfides.
In such cases we are trying to avoid adding the reducing agents. 

I have also seen many examples of mesophilic bacterial proteins and mammalian 
proteins forming disulfides
when overexpressed in E.coli. Sometimes to get homogeneous sample we add 
disulfide enforcing agent
(diamide) as early as induction stage when overexpressing in E.coli. 

Best wishes,

Misha

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat 
[rkha...@ccny.cuny.edu]
Sent: 20 December 2014 19:05
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] non-specific disulfide bonds in crystal structures

Hi Todd,

A concern of mine is that you may be looking at an artifact
of the domain. Whatever assays you decide to do, make sure
that they are also performed for the full sized protein.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Sat, 20 Dec 2014 10:44:25 -0800
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on
behalf of Christine Gee chr...@gmail.com)
Subject: Re: [ccp4bb] non-specific disulfide bonds in
crystal structures
To: CCP4BB@JISCMAIL.AC.UK

   Hi Todd,
   I used to work on PNMT which also is supposedly
   monomeric and formed a disulfide between monomers in
   the crystals.
See
http://www.sciencedirect.com/science/article/pii/S157096390
5000968?via=ihub
   We showed that it was irrelevant to activity.
   Cheers
   Christine
   Sent from my iPad
   On 20 Dec 2014, at 9:52 am, Todd Jason Green
   tgr...@uab.edu wrote:

 Hello All-

 I have recently determined a domain structure of a
 larger protein. The structure shows a clear
 disulfide bond between two monomers in the
 asymmetric unit. I'm trying to figure out if this
 is an artifact of the crystal packing or has
 biological relevance. The protein has been
 reported to function as a monomer. If I look at
 the pool of protein on a SDS-PAGE gel under
 non-reducing conditions, I see that a smaller
 percentage (~15-20%) of the protein runs as a
 dimer. In the structure, the association has
 2-fold symmetry with about 29% of the monomeric
 surface area buried between the dimer. Can anyone
 point me in the direction of a paper describing a
 non-specific disulfide in a crystal, or perhaps a
 criteria for assessing specificity? I will do some
 functional studies, but I'm looking for some info
 on a lazy saturday.

 Thanks in advance. Best-
 Todd

Re: [ccp4bb] I222 - P22121 space-group ambiguity

[Isupov, Michail]

Hi Florian,

When your pseudo-translation (native Patterson peak) is close to (0.5,0.5,0.5) 
even in p22121 you will have have most of reflections with
h+k+l=2n+1 
measured as weak. In your lower resolution datasets 2.6 and 3.0 A non-weak 
reflections can be lost in noise and your group assignment
may go wrong, and all of your crystals may be P22121.

Having said this, polymorphism is quite common.
In a recent publication Acta Crystallogr D Biol Crystallogr. 2014 Sep 1;70(Pt 
9):2430-43, Lebedev  Isupov
we describe a case where two crystals had similar cell parameters in different 
space groups

a = 119.2, b = 192.5, c = 77.3 P212121
and
a = 112.0, b = 192.2, c = 76.7 P21212.

Both structures could be refined in the other space group at 1.8A resolution, 
but resulting R-frees were 8-15 % higher in the wrong space group.
Both space group appeared as a result of symmetry breakdown in a supergroup 
A2122 (reindexed C2221).
P212121  structure could also be refined in the supergroup with FreeR only 3 % 
higher than in the real space group.
However as half of reflections had to be thrown out to reindex to A2122 even if 
FreeR would be equal in the supergroup and P212121
I would consider the latter correct.

I would recommend to  solve and refine your low resolution structures in all 
possible space groups and choose the one with  better refinement statistics,
preferring  not centered one in case of equal Rfrees.

Quality of density maps could be criteria in borderline cases.

Cheers,

Misha



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Florian 
Schmitzberger [schmitzber...@crystal.harvard.edu]
Sent: 13 October 2014 09:47
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] I222 - P22121 space-group ambiguity

Hi everybody,

I collected a number of X-ray data sets from crystals originating from the same 
cryst. drop. I solved the initial structure in P22121 space group by MR with 
Phaser locating two molecules (data to ~ 2.1 Angstr.); refined R/Rfree: 
0.213/0.244.

Processing of some of the other data sets with XDS/Aimless is consistent with 
I222 space group (resolution ~ 2.6 Ang.). I can locate one molecule. The 
unit-cell dimensions for I222 and the initial P22121 space groups for two of 
the data sets are:
I222: a=87.8 b=101.18 c=123.63; P22121: a=93.34 b=105.47 c=122.98;

I superposed the molecule in I222 onto one of the two located for the initially 
solved P22121; the orientation of the NCS-related molecule in P22121 differs 
from the crystallographic-symmetry related one in I222. Trying to solve this 
P22121 data set in I222 with MR, does not result in high Z scores, and maps do 
not look good.

Some of the data sets that process in I222 to ~ 3 Angstr., I can also solve in 
P22121, locating two molecules (differences may not be that clear in this case, 
since the resolution is lower).

Some other data sets process in P22121 with Aimless; with a substantial 
off-origin Patterson peak, indicating translational NCS. For these, Phaser 
positions two molecules that are related by crystallographic translational NCS. 
These two molecules are crystallographic-symmetry related in the original 
P22121 data set. I can also solve these data sets in I222 space group, with the 
overall Z score higher than for the P22121 data.

I am uncertain, what the ‘true’ space group for some of my data sets is. Could 
it be that for data that process in P22121, but can be solved in I222, 
reflections that would indicate I222 space group were not collected? 
Alternatively, perhaps what I am seeing is that there is a (gradual) transition 
of the crystal lattice (between P22121 and I222 or vice versa), caused by 
variation in crystal handling/cooling or exposure to X-rays.

It’s relevant to me, because in P22121 space group, a region of the molecule 
that is of biological interest makes NCS-related crystal contacts that are 
crystallographic-symmetry related in I222.

Has anybody observed similar cases? I would appreciate comments.

Cheers,

Florian

Re: [ccp4bb] Proper detwinning?

[Isupov, Michail]

I would recommend to run ZANUDA in the default mode from ccp4i or on CCP4 web 
server.
ZANUDA has resolved several similar cases for me.

Misha


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage 
[cdf...@gmail.com]
Sent: 10 July 2014 01:14
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Proper detwinning?

Hi Everyone,

Despite modelling completely into great electron density, Rwork/Rfree
stalled at ~38%/44% during refinement of my 2.0-angstrom structure
(P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning,
with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447.
However, there are no twin laws in this space group. I reprocessed the
dataset in P21 (8 monomers/AU), which did not alter Rwork/Rfree, and
in P1 (16 monomers/AU), which dropped Rwork/Rfree to ~27%/32%. Xtriage
reported the pseudo-merohedral twin laws below.

P21:
h, -k, -l

P1:
h, -k, -l;
-h, k, -l;
-h, -k, l

Performing intensity-based twin refinement in Refmac5 dropped
Rwork/Rfree to ~27%/34% (P21) and ~18%/22% (P1). Would it be
appropriate to continue with twin refinement in space group P1? How do
I know I'm taking the right approach?

Interestingly, I solved the structure of the same protein in P212121
at 2.8 angstroms from a different crystal. Rwork/Rfree bottomed out at
~21%/26%. One unit cell dimension is 9 angstroms greater in the
twinned dataset than in the untwinned.

Thank you for any suggestions!

Regards,
Chris

Re: [ccp4bb] Fwd: Twinning

[Isupov, Michail]

Dear Steve,

Presence of several Patterson peaks at impossible (as far as crystal packing is 
concerned)  distances
and an NCS two-fold normal to the crystallographic one suggests it could
be  an order-disorder (OD) structure, like the one we had in Exeter.

An order-disorder twin crystal of L-2-haloacid dehalogenase from Sulfolobus 
tokodaii.
Rye CA, Isupov MN, Lebedev AA, Littlechild JA.
Acta Crystallogr D Biol Crystallogr. 2007 Aug;63(Pt 8):926-30.


For that case at 1.6 A Andrey Lebedev from York had written a dedicated 
detwinning (demodulation) program
and FreeR went down from 0.27 to 0.22. Original FreeR was nearly acceptable as 
only several percent of reflections
were overlapping.
If you feel your case appears similar to our OD-structure I would suggest you 
to contact Andrey, as he is collecting
cases of OD-structures and probably could modify his program for your case.

Best wishes

Misha Isupov
University of Exeter

From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Stephen Hare 
[s.h...@mail.cryst.bbk.ac.uk]
Sent: 06 January 2009 16:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fwd: Twinning

Dear All,

We are currently working on a structure of apparent P21 symmetry which
has been solved by molecular replacement. The data are to 2.7Å but the
Rfree will not drop below 30%. The density is clear for the model we
have, however there is extra density that suggests a shift of the
structure by 16Å in either direction - resulting in three possible
overlapping positions for the structure. We assume this is the result
of twinning.

The unit cell dimensions are 102.7Å, 83.0Å, 115.3Å, 90°, 101.8°, 90°.
Examining the data with phenix.xtriage also suggests pseudo
translational symmetry with a separation of 16Å.  A Patterson peak at
0.097, 0.000, -0.096 is approximately 30% of the origin peak, while a
second peak of double the translation at 0.192, 0.000, 0.195 is 7% of
the origin peak. The structure contains a dimer of dimers with an NCS
2 fold axis almost perpendicular to the crystallographic 2 fold. This
NCS axis almost  coincides with the diagonal between the A and C axes.
A twin axis along the A C diagonal (l,-k,h) could explain the observed
extra density, however this is not possible because A and C are
different lengths.

As a result of the NCS axis running almost perpendicular to the
observed P21 axis, it is possible to merge the reflections in a larger
orthorhombic unit cell - dimensions 137.1Å, 83.3Å, 169.8Å although
here the Rmerge is higher and it is not possible to get a molecular
replacement solution.

Is it possible to define the (l,-k,h) twinning operator in our
original unit cell? or have we missed the actual unit cell?
Orsomething else?

Steve



Stephen Hare PhD
Post doctoral research associate
Jefferiss Research Laboratories
Wright-Fleming Institute
Division of Medicine
Imperial College London
Norfolk Place
London W2 1PG
UK

Phone: +44 (0) 20 7594 3908
Fax: +44 (0) 20 7594 3906

Re: [ccp4bb] twin axis in p21

[Isupov, Michail]

Dear Hua,

Your problem sounds like a 'false origin' one, where due to the 
pseudotranslation the molecular replacement
choses the wrong origin out of the two possible ones. Your space group is 
probably P212121.
The best approach is to run Andrey Lebedev's program ZANUDA on YSBL server.

http://www.ysbl.york.ac.uk/YSBLPrograms/index.jsp


Best wishes

Misha Isupov


From: CCP4 bulletin board [EMAIL PROTECTED] On Behalf Of Yuan, Hua [EMAIL 
PROTECTED]
Sent: 26 November 2008 01:20
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] twin axis in p21

Dear Colleagues,

I'd like to share with you with a twinning puzzle that's been
troubling me for a while.
I have a ~2.2 A data merged in p212121 space group with unit cell:
41.8300  117.4490  134.4840   90.   90.   90.
Translational NCS was detected but structure solution was able to be
obtained by phased molecular replacement as we have a Au derivative
data set. Obtaining experimental phase or MR alone didn't work.

However, with this model and data set, the refinement didn't work
(with R-free ~40% or higher if with more cycles).  Different twinning
tests have been carried out and no sign of twinning.  All possible
p222 space groups have been tried but no luck or even worse.
Considering that translational symmetry and perfect twinning may make
things deceiving, the data was merged into p21 and a twin-refinement
was performed in phenix.refine.  After 3 cycles of rigid-body and
group ADP, a R-free of ~30% was obtained and the output map looks OK.
It looks like we got it.  But then things get interesting when we
tested other two axis as the p21 unique axis.  With all the three
choices plus corresponding twin-refinement, we could get a similar
R-free (30%).  Besides, the three maps all look OK except for one
missing some side chains.

Then the structure was solve in p1 and Xtriage was used with the hope
that one axis would stand out.  However, the RvsR for all three axis
are almost the same.  Did I miss anything here?  Of course, we could
pick up the one axis we like best and get the structure solved ^---^.
However, this puzzle would eat me alive for a very long time.

Best,

Hua