Re: [ccp4bb] To Trim or Not to To Trim

[benjamin bax]

Hi,
Probably a stupid question. 
Could you multiply a, b and c cell dimensions by 2 or 3 (to give 8 or 27 
structures) and restrain well defined parts of structure to be ‘identical’ ? To 
give you a more NMR like chemically sensible ensemble of structures?
Ben


> On 18 Mar 2023, at 12:04, Helen Ginn  wrote:
> 
> Models for crystallography have two purposes: refinement and interpretation. 
> Here these two purposes are in conflict. Neither case is handled well by 
> either trim or not trim scenario, but trimming results in a deficit for 
> refinement and not-trimming results in a deficit for interpretation.
> 
> Our computational tools are not “fixed” in the same way that the standard 
> amino acids are “fixed” or your government’s bureaucracy pathways are 
> “fixed”. They are open for debate and for adjustments. This is a fine example 
> where it may be more productive to discuss the options for making changes to 
> the model itself or its representation, to better account for awkward 
> situations such as these. Otherwise we are left figuring out the best 
> imperfect way to use an imperfect tool (as all tools are, to varying 
> degrees!), which isn’t satisfying for enough people, enough of the time.
> 
> I now appreciate the hypocrisy in the argument “do not trim, but also don’t 
> model disordered regions”, even though I’d be keen to avoid trimming. This 
> discussion has therefore softened my own viewpoint.
> 
> My refinement models (as implemented in Vagabond) do away with the concept of 
> B factors precisely for the anguish it causes here, and refines a 
> distribution of protein conformations which is sampled to generate an 
> ensemble. By describing the conformations through the torsion angles that 
> comprise the protein, modelling flexibility of a disordered lysine is 
> comparatively trivial, and indeed modelling all possible conformations of a 
> disordered loop becomes feasible. Lysines end up looking like a frayed end of 
> a rope. Each conformation can produce its own solvent mask, which can be 
> summed together to produce a blurring of density that matches what you would 
> expect to see in the crystal.
> 
> In my experience this doesn’t drop the R factors as much as you’d assume, 
> because blurred out protein density does look very much like solvent, but it 
> vastly improves the interpretability of the model. This also better models 
> the boundary between the atoms you would trim and those you’d leave 
> untrimmed, by avoiding such a binary distinction. No fear of trimming and 
> pushing those errors unseen into the rest of the structure. No fear of 
> leaving atoms in with an inadequate B factor model that cannot capture the 
> nature of the disorder.
> 
> Vagabond is undergoing a heavy rewrite though, and is not yet ready for human 
> consumption. Its first iteration worked on single-dataset-single-model 
> refinement, which handled disordered side chains well enough, with no need to 
> decide to exclude atoms. The heart of the issue lies in main chain 
> flexibility, and this must be handled correctly, for reasons of 
> interpretability and elucidating the biological impact. This model isn’t 
> perfect either, and necessitates its own compromises - but will provide 
> another tool in the structural biology arsenal.
> 
> —-
> 
> Dr Helen Ginn
> Group leader, DESY
> Hamburg Advanced Research Centre for Bioorganic Chemistry (HARBOR)
> Luruper Chaussee 149
> 22607 Hamburg
> 
> 
> 
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Re: [ccp4bb] TWIN?

[benjamin bax]

Hi, 
  I have had twinning with P61, which makes it look like P6122. This is with 
slightly asymmetric dimer. 
You could try mol replacement in P65, and refine with twinning on. Maps should 
look better if correct. Ben 

Sent from my iPhone

> On 9 Jun 2021, at 10:13, Randy John Read  wrote:
> 
> I agree with Kay that, with a good model, solving in P1 is likely to be the 
> easiest comprehensive solution.  If that doesn’t work, Phaser starts every 
> MR_AUTO job by making a list of all the subgroups, including the different 
> potential indexings (represented by Hall symbols), so you could also work 
> through those systematically.  You provide a “SPACEGROUP HALL” command with 
> one of the Hall symbols for each possibility, and Phaser will expand the data 
> from the higher symmetry and reindex as required.
> 
> Best wishes,
> 
> Randy Read
> 
>> On 9 Jun 2021, at 09:37, Kay Diederichs  
>> wrote:
>> 
>> Hi Almudena,
>> 
>> if it is a packing problem, you need to find the correct subgroup of P6522 
>> (179).
>> Take a look at 
>> https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups
>>  .
>> Subgroups of 179 are 20, 153, 154, 170, and recursively each of these has 
>> subgroups:
>> 20 has  4 and 5 as subgroups, 153 and 154 both have 5 and 145 as subgroups, 
>> 170 has  4 and 145 as subgroups - taken together, 4, 5 and 145.
>> These have 1 as subgroup.
>> So the true space group could be P1, P21, C2, P32, C2221, P3212, P3221, P65.
>> You could run molecular replacement with all of these. Unfortunately, there 
>> may be several ways to index the data in some of these space groups, and 
>> they may not be equivalent. For example, I think there are 3 non-equivalent 
>> ways to index in C2 or P21 if the Laue class is 6/m .
>> 
>> If there is twinning, the intensity statistics should tell about that - but 
>> they may be set off by tNCS. 
>> 
>> One way to overcome the mess of possible space groups and settings plus the 
>> twinning possibility is to index and solve the structure in P1. That should 
>> allow a packing without clashes, and one could identify the correct space 
>> group by running POINTLESS on the Fcalc, and/or Zanuda. Since you have a 
>> good model, I'd try that.
>> 
>> Hope this helps,
>> Kay
>> 
>>> On Tue, 8 Jun 2021 17:14:30 +0200, Almudena Ponce Salvatierra 
>>>  wrote:
>>> 
>>> Hello everyone,
>>> 
>>> I am working with an RNA-only structure, data are at 3 Angstroms, and at
>>> first, I thought was in the C2 space group (with 6 molecules in the AU).
>>> 
>>> I can't finish building! it, because the structure seems to get in the way
>>> of its neighbor symmetrically! See the attached picture, please! The only 4
>>> residues that the structure is missing "have to go there", where one
>>> structure meets the other one. The R factors for this spacegroup are around
>>> 0.3.
>>> 
>>> However, Phenix Xtriage suggests the symmetry may be higher. So I reindex
>>> in P622, do MR with Phaser (trying all possible space groups in that point
>>> group), and it finds a unique solution in space group P 65 2 2 with TFZ of
>>> 50 and LLG >3000. Of course, the problem persists (one molecule sort of
>>> interfering with the neighbor), not only that but also the refinement
>>> R-factors are substantially higher, 0.37.
>>> 
>>> I have to say that the refinement maps look better when I am working with
>>> the C2 spacegroup. I can't understand, though, what is happening at that
>>> "supposedly interface" between the two molecules. Has anybody experienced
>>> anything like this in the past? Can it be a twin? Is it something else?
>>> and... how to fix it?
>>> 
>>> Thank you very much in advance.
>>> 
>>> Best wishes,
>>> 
>>> Almudena
>>> 
>>> 
>>> 
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>> 
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> 
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research Tel: +44 1223 336500
> The Keith Peters Building   Fax: +44 1223 336827
> Hills Road   E-mail: 
> rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K. 

Re: [ccp4bb] superimposition of 3D structures with the DNA part only

[benjamin bax]

Hi Eleanor,
   Thanks for fix.

Sometimes I just superpose three atoms (making sure they are not in a straight 
line).
But my atom superposition lsqkab script (below) - suggests that lsqkab has a 
different atom count than used ‘standardly’. 
Has anyone else found this problematic?

Thanks, Ben 
  

cat lsq_compound_new.com 
#!/bin/sh 

set -e
# 
# nb xyzin1 is reference structure - not moving. 
lsqkab xyzin2 ABITIO-p-nh-p-nometal.pdb xyzin1 1S16-on_mola_ndom.pdb \
XYZOUT abit_nomet_on_1s16.pdb  \
RMSTAB sub_suba_rms.tab  < wrote:

I MEANT to upgrade lsqkab to accept DNA, and there is a small possibility that 
I did!
Cheers Eleanor

On Tue, 28 Apr 2020 at 11:52, Carter, Charlie mailto:car...@med.unc.edu>> wrote:
In my experience, lsqkab wouldn’t orient nucleic acid atoms, and I think 
Eleanor once told me I needed a different alternative for nucleic acids. If 
this is no longer true, I’m happy to learn of it.

Charlie
> On Apr 28, 2020, at 6:40 AM, benjamin bax  <mailto:ben.d.v@gmail.com>> wrote:
> 
> 
> HI Fred, 
> 
>   I still use command line version of lsqkab to do this kind of DNA fitting - 
> script below only uses mainchain atoms (not bases) which helps if you have 
> different DNAs.
> Chain E and F are DNA. 
> 
>Ben 
> 
> ./lsq-hinge-6fqv-bin-EV-B.com <http://lsq-hinge-6fqv-bin-ev-b.com/> > 
> lsq-hinge-6fqv-bin-EV-B.log
> 
> 
> cat lsq-hinge-6fqv-bin-EV-B.com <http://lsq-hinge-6fqv-bin-ev-b.com/> 
> #!/bin/sh 
> 
> set -e
> # Obtain NCS rotation/translation relating CHAIN R to CHAIN S
> # 
> # nb xyzin1 is reference structure - not moving. 
> lsqkab xyzin2 ./6fqv-binary-B-on-2XCS-ToprimB.pdb xyzin1 ./2xcs-c1a.pdb \
> XYZOUT binaryB-on-3-6E-15-18F.pdb  \
> RMSTAB test1.tab  < # 
> #  DNA fit two strands - trying for just backbone. 
> # 
> FIT RESIDUE MAIN 3 TO 6 CHAIN E
> MATCH RESIDUE MAIN 3 TO 6 CHAIN E
> FIT RESIDUE MAIN 15 TO 18 CHAIN F
> MATCH RESIDUE MAIN 15 TO 18 CHAIN F
> OUTPUT  RMS# ! output file RMSTAB with differences
> OUTPUT  XYZ# ! output file RMSTAB with differences
> END
> EOF
> 
> On 24 Apr 2020, at 12:08, Fred. Vellieux  <mailto:frederic.velli...@lf1.cuni.cz>> wrote:
> 
> Hi folks,
> 
> Some of you may have had to do this already. Either in the lab or more 
> recently perhaps from home.
> 
> I have two structures that I wish to superpose (two protein:dsDNA complexes). 
> Not using the protein part, but superposition through the dsDNA.
> 
> I'm not quite certain what is the "best" way of doing this.
> 
> Your suggestions will be appreciated, thanks.
> 
> Fred. Vellieux
> 
> -- 
> MedChem, 1st F. Medicine, Charles University
> BIOCEV, Vestec, Czech Republic
> 
> 
> 
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Re: [ccp4bb] superimposition of 3D structures with the DNA part only

[benjamin bax]

HI Fred, 

  I still use command line version of lsqkab to do this kind of DNA fitting - 
script below only uses mainchain atoms (not bases) which helps if you have 
different DNAs.
Chain E and F are DNA. 

   Ben 

./lsq-hinge-6fqv-bin-EV-B.com > lsq-hinge-6fqv-bin-EV-B.log


cat lsq-hinge-6fqv-bin-EV-B.com 
#!/bin/sh 

set -e
# Obtain NCS rotation/translation relating CHAIN R to CHAIN S
# 
# nb xyzin1 is reference structure - not moving. 
lsqkab xyzin2 ./6fqv-binary-B-on-2XCS-ToprimB.pdb xyzin1 ./2xcs-c1a.pdb \
XYZOUT binaryB-on-3-6E-15-18F.pdb  \
RMSTAB test1.tab  < wrote:

Hi folks,

Some of you may have had to do this already. Either in the lab or more recently 
perhaps from home.

I have two structures that I wish to superpose (two protein:dsDNA complexes). 
Not using the protein part, but superposition through the dsDNA.

I'm not quite certain what is the "best" way of doing this.

Your suggestions will be appreciated, thanks.

Fred. Vellieux

-- 
MedChem, 1st F. Medicine, Charles University
BIOCEV, Vestec, Czech Republic



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[ccp4bb] REMARK 280 in mmcif?

[benjamin bax]

In PDB files you could put all information about protein delivery buffer, 
crystallisation conditions and cryo conditions in
REMARK 280

(and pull it through in refmac with keyword
pdbout copy remarks 280
)

Where are you meant to put all the information about what could be in your 
crystal in mmcif_pdbx?

Thanks, Ben 

Web (below) does not seem to give any very clear answers and I could not work 
it out from looking at half a dozen recent pdb entries.
Data item below is used in zero depositions. .. 


Data items in the PDBX_EXPTL_CRYSTAL_GROW_COMP category record
   details about the components of the solutions that were 'mixed'
   to produce the crystal.
   
http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v40.dic/Categories/pdbx_exptl_crystal_grow_comp.html

loop_
_pdbx_exptl_crystal_grow_comp.crystal_id
_pdbx_exptl_crystal_grow_comp.sol_id
_pdbx_exptl_crystal_grow_comp.comp_id
_pdbx_exptl_crystal_grow_comp.comp_name
_pdbx_exptl_crystal_grow_comp.conc
_pdbx_exptl_crystal_grow_comp.conc_range
_pdbx_exptl_crystal_grow_comp.conc_units
4'protein' 1  'protein'   25..  'mg/ml'
4'protein' 2  'Tris HCl'  20..  'millimolar'
4'protein' 3  'NaCl'   0.2   .  'molar'
4'precipitant' 1  'PEG 4000'  12.5   .  'percent_weight_by_volume'
4'precipitant' 2  'MES'0.1   .  'molar'
pdbout copy remarks 280


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

[benjamin bax]

Hi Katherine,

One possibility could be that you have occupancy of 0.5 and that ligand 
binding at one site in the crystal distorts a second site so it cannot bind 
ligand.
Occupancy refinement is not always very stable. 
If you are looking at two structures on top of one another - refinement needs 
to be done carefully.
One thing I sometimes try is setting occupancy at various levels for the two 
different conformations you have modelled, e.g. 0.2/0.8, 0.3/0.7, 0.4/0.6, 
0.5/0.5, 0.6/0.4, 0.7/0.3, 0.8/0.2
Then check all maps carefully. 
 
 Sometimes soaking a crystal can change space-group and you may need to 
reprocess data in lower symmetry cells to check. If you have only one molecule 
in asymmetric unit might be worth trying the three P21 cells
(with beta=90 degrees). 

[However, if you have to use DMF (rather than DMSO) to get your ligand into 
solution - I would ask your chemists to make/give you some more soluble 
analogues. ]

  Ben Bax 


Ben Bax
 
Reader in Structural Biology
Medicines Discovery Institute
Cardiff University
Main Building
Park Place
Cardiff
CF10 3AT 
 
Email: b...@cardiff.ac.uk 

Ben Bax
 
Darllenydd mewn Bioleg Strwythurol
Sefydliad Darganfod Meddyginiaethau
Prifysgol Caerdydd
Prif Adeilad
Plas y Parc
Caerdydd
CF10 3AT
 
Ebost: b...@cardiff.ac.uk


On 20 Dec 2019, at 04:57, Katherine Lim  
wrote:

Hi all,

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

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

Space Group P212121
Unit cell abc   84.76, 89.84, 91.55
unit cell alpha beta gamma  90, 90, 90

OVERALL LOW RES HIGH RES
Low res limit   45.78   45.78   1.94
High res limit  1.9 9.111.9
Rmerge  0.234   0.052   1.684
Rmeas   0.244   0.054   1.768
Rpim0.069   0.016   0.527
Total # observations663073  628236851
Total # unique  55174   579 3359
I/sigma 7.5 27.91.4
CC ½0.997   0.999   0.747
Completeness %
99  98.794.7
Multiplicity12  10.811
Katherine Lim 


PhD Candidate

School of Biomedical Sciences; School of Molecular Sciences

Marshall Centre for Infectious Disease Research and Training 

The University of Western Australia 
  


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Re: [ccp4bb] photograph of Enraf-Nonius FAST detector

[benjamin bax]

Hi Harry,
  I think some old stuff from Birkbeck crystallography teaching labs. went to 
science museum.

However, picture of 
Enraf-Nonius Weissenberg X-ray camera, model Y809, (X-ray diffraction camera) - 
from 1968 - does not look quite like what I remember.

https://collection.sciencemuseum.org.uk/search?q=Enraf-Nonius%20Weissenberg%20X-ray%20camera%2C%20model%20Y809%2C%20(X-ray%20diffraction%20camera)

Ben



On 27 Nov 2018, at 12:48, Harry Powell 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:

Hi Elspeth

That's brilliant - no, it's not too late by any means. Photos of other obsolete 
detectors are also welcome!


On 27 Nov 2018, at 12:40, Elspeth Garman wrote:

> I have one of the LMB Cambridge one. 
> Can dig it out for you but not till Monday. Is that too late?
> Elspeth 
> 
> Sent from my iPhone
> 
> On 27 Nov 2018, at 12:35, Harry Powell 
> <193323b1e616-dmarc-requ...@jiscmail.ac.uk 
> > wrote:
> 
>> Hi
>> 
>> I was wondering if anyone out there has a decent-quality photograph of an 
>> Enraf-Nonius FAST detector that I could use? I believe that one was 
>> installed at SRS Daresbury in 1983...
>> 
>> Harry
>> --
>> Dr Harry Powell
>> 


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[ccp4bb] Fwd: Senior Laboratory Technician - Protein Purification - in the Medicines Discovery Institute at Cardiff University.

[benjamin bax]

We are advertising for a Senior Laboratory Technician Protein Purification in 
the Medicines Discovery Institute at Cardiff University, UK.
 
The goal of the newly-established Medicines Discovery Institute in Cardiff is 
to translate world-leading research on scientific understanding of disease 
mechanisms into innovative therapeutic approaches.  Our vision is to play a 
leading role in discovering new medicines in areas of high unmet need, with a 
particular emphasis on, but not exclusive to, neuroscience and mental health.
 
Closing date: Sunday, 15 July 2018.
 
Interested candidates please apply via University of Cardiff job page:
https://krb-sjobs.brassring.com/TGnewUI/Search/home/HomeWithPreLoad?PageType=JobDetails&partnerid=30011&siteid=5460&AReq=7452BR#jobDetails=1239864_5460
 

  
Informal inquiries to:  b...@cardiff.ac.uk  

 





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Re: [ccp4bb] query of B chain model of protein

[benjamin bax]

Hi,
  Looks like you have some density for same conformation as in A chain.

It could be you have two (or more) conformations for some parts of the 
structure in MolB. It is often tricky to sort out when you have two 
conformations for residues and map is ambiguous (and at 2.8A). 

I would rigid body fit chunks of residues (6-10 residues?) that have density in 
difference map - then prune back things with no density in difference map 
(delete residues and atoms on side-chains outside of density - with delete atom 
in coot).

 Then set occupancy of ‘new’ stuff to 0.3 and do a little tightly restrained 
refinement and calculate new maps (in my experience - if you set occupancy to 
0.5 you are likely to get too much model bias - to see other conformation). 
Quite likely this will not help and it will still be ambiguous. 

[ Alternatively - If you solve another 50 structures of the same thing - in 
different space-groups/cells - you may find different conformations for the 
'missing parts’ appear in a couple of structures - and then you may be able to 
better model them in MolB of this structure]. 

  Ben
 

On 2 Oct 2017, at 15:42, Vikram Dalal  wrote:

Hi all,


I am solving a protein data of 2.6 A. It has two monomers in ASU. It has 377 
residues. I have built residues 26 to 36 and 41 to 377 in A chain while B chain 
has model 42 to 129, 146 to 168 and 189 to 371. Current R factor and R Free is 
23.2  and 28.5, respectively. I have attached electron density (fourier map at 
1.0 contour and difference map at 2.5) figures for 130 to 145, 169 to 188 and C 
terminal region of B chain and superposed model of A chain is shown in green 
color. 

I have tried manually building missing residues of B chain according to A chain 
model, but R free increased to 29. 

Any suggestions will be highly aprreciated. 


Thanks & Regards,







Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] Incorrect Structure in the PDB

[benjamin bax]

Hi Trevor,
  Some of my colleagues came across a misinterpreted structure published in 
Acta D., a couple of years ago.
They raised the issue with the editors of Acta D., who were most helpful in 
getting the issue sorted out.

My impression is that the journals are the responsible for the papers that they 
publish, and raising this type of issue with the journal editors initially - 
may be the best way forward.

Best regards, Ben 


On 27 Jun 2017, at 08:15, Trevor Sewell  wrote:

The misinterpretation is considerable I as can be seen from the attached coot 
screenshot.
 
I have no reason to suspect malfeasance. But it looks like the authors didn’t 
check very carefully.
I have re-interpreted and refined the density and it is just fine – Rfactor of 
18% for a 2.3A structure.
 
The critical reinterpretations concern  the orientation of the backbone near 
the active site and the interpretation of a blob of density claimed to be 
substrate in the original paper.
 
Maybe the best would be to write to the author and suggest that she obsolete 
the structure. We could see if we could  reach some agreement on how to take it 
further – perhaps a letter to the editor of JBC.
 
 
 
Sent from Mail  for Windows 10
 
From: Manfred S. Weiss 
Sent: Tuesday, June 27, 2017 8:46 AM
To: Trevor Sewell 
Subject: Re: [ccp4bb] Incorrect Structure in the PDB
 
Dear Trevor,

you can download the incorrect structure and the associated data and 
reinterpret and
re-refine the structure. Then you can re-deposit provided you write a paper 
about the
new findings. This is currently the policy of the PDB.

Else, you can contact the authors of the incorrect structure and do the 
reinterpretation
together with them? They can replace the incorrect structure without a new 
publication.

That's all there is at the moment.

May I ask what is incorrect about the structure?

Cheers, 

Manfred

Am 27.06.2017 um 08:34 schrieb Trevor Sewell:
>  
> I have come across a key paper in my field that describes an enzyme 
> mechanism. Their work is based on a deposited structure – by other authors - 
> that is incorrectly interpreted.
>  
> Is there a process for removing a demonstrably wrong structure (deposited by 
> others) from the PDB and replacing it with a correctly interpreted structure 
> based on the original data? Or is there an alternative, and generally 
> recognized, way of getting the correct structure in the public domain? 
>  
> Many thanks for your advice on this matter.
>  
> Trevor Sewell
>  
> Disclaimer - University of Cape Town This e-mail is subject to UCT policies 
> and e-mail disclaimer published on our website 
> athttp://www.uct.ac.za/about/policies/emaildisclaimer/ 
>  or obtainable from +27 21 
> 650 9111. If this e-mail is not related to the business of UCT, it is sent by 
> the sender in an individual capacity. Please report security incidents or 
> abuse via cs...@uct.ac.za 
-- 
Dr. Manfred S. Weiss
Macromolecular Crystallography
Helmholtz-Zentrum Berlin
Albert-Einstein-Str. 15
D-12489 Berlin
Germany


Helmholtz-Zentrum Berlin für Materialien und Energie GmbH

Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V.

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Jutta Koch-Unterseher
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Disclaimer - University of Cape Town This e-mail is subject to UCT policies and 
e-mail disclaimer published on our website at 
http://www.uct.ac.za/about/policies/emaildisclaimer/ 
 or obtainable from +27 
21 650 9111. If this e-mail is not related to the business of UCT, it is sent 
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abuse via cs...@uct.ac.za 


Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] What are acceptable Rwork/Rfree for publication

[benjamin bax]

Hi Khoa,

  How many rounds of refine and rebuild have you gone through on the graphics?

Have you tried Lorestr in CCP4 (Automated refinement of macromolecular 
structures at low resolution using prior information, Oleg Kovalevskiy, Robert 
A. Nicholls and Garib N. Murshudov). 

Ben 

[At 2.9A resolution traditionallly I would calculate maps with refmac, phenix 
and buster with and without tls refinement.
Then go through the structure looking at all maps and rebuilding. 
Information content at 2.9A is sometimes limited and structural changes only 
appear slowly with careful refinement (5-10 rounds).]


On 14 Jun 2017, at 14:09, Khoa Pham  wrote:

Dear CCP4 members,

I am refining a structure with a resolution of ~ 2.9 A and the Rwork/Rfree are 
are 0.34/0.37, respectively. 
My question is that these Rwork/Rfree are acceptable for publication. If not, 
how can I reduce them?

Thank you.

Khoa

The table of statics.
Space group
C121

Resolution (Å)
29.69-2.92
No of unique reflections
61569 (8747)
Rmerge (%)
20 (350)
Rpim (%)
11 (198)
I/s(I)
8.3 (0.5)
CC1/2
0.99 (0.33)
CCanomalous
N/A
Completeness (%)
98.5 (95.8)
Redundancy
4.2 (4.0)
 
 
Refinement
 
Resolution (Å)
29.69-2.92
No of reflections
58394 (4069)
Rwork/Rfree
0.3380/0.3689

R.m.s. deviations
 
   Bond lengths (Å)
0.0073
   Bond angles (o)
1.1062


Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] Conserved water

[benjamin bax]

How about 
WONKA and OOMMPPAA: analysis of protein–ligand interaction data to direct 
structure-based drug design?
Ben 



On 7 Jun 2017, at 07:59, Eleanor Dodson  wrote:

Many years ago I wrote code to label waters with a code related to the 
residue/atom  they were Hbonded to , so then you could check whether all OH TYR 
227 in each chain  had an associated water.. But it used non-standard water 
naming ..

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.

Eleanor

On 7 June 2017 at 00:30, gerardo andres 
<130afa955101-dmarc-requ...@jiscmail.ac.uk 
> wrote:
Hi everyone, does anyone know any strategy or program (besides pywater) to 
identify conserved waters in a protein?

Thanks, 

Gerardo



Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] CH-bond length discrepancies

[benjamin bax]

'Most   distances   between bonded  atoms   weresettled longago 
to  highaccuracy,   but,in  the caseof  
hydrogens,  the values  in  common  use often   differ  by  
as  muchas  20%.'

Phenix  /   MolProbity  HydrogenParameter   Update

Deis, L. N., Verma, V., Videau, L. L., Prisant, M. G., Moriarty, N. W.,

Headd, J. J., Chen, V. B., Adams, P. D., Snoeyink, J., Richardson, J. S. & 
Richardson, D. C. (2013). Comput. Crystallogr. Newsl. 4, 9–10. 



On 28 Apr 2017, at 18:33, Bernhard Rupp  wrote:

Dear Fellows of the Bond,
 
when validating a QM refined homology model with Molprobity, I noticed various 
8 sigma deviations in the carbon-hydrogen bond distances.
Out of curiosity, I then used refmac to calculate riding Hs for the same model, 
and at least in one instance (N-H backbone) there are 
significant differences between Molprobity and Refmac H bond distances 
(differences to the QM distances in other 
instances I find interesting, but less relevant for us).
 
The riding H vs Molprobity presumably should be consistent, because if we use 
them in VDW restraints but
they differ from the validation target, systematic bias will occur. I have no 
feel how significant that effect
might be – maybe someone more erudite can comment.
 
Examples
 
distance  MP   REF QM
backbone N-H   0.861.011.00   
phenyl C-H 0.930.931.09
 
Best, BR
 
PS: If someone has accurate experimental values for CH distances I’d appreciate 
a link.
No access to CSD.
 
--
Bernhard Rupp
Crystallographiae Vindicis Militum Ordo
http://www.hofkristallamt.org/ 
b...@hofkristallamt.org 
+1 925 209 7429
+43 767 571 0536
--
Many plausible ideas vanish 
at the presence of thought
--


Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] Ligand electron density

[benjamin bax]

Hi,
   A think a detailed discussion is off topic for CCP4 bb.
https://www.quantiki.org/wiki/quantum-sock-theory
  Ben 

On 2 Apr 2017, at 00:31, Phoebe A. Rice  wrote:

At long last, a brilliant theoretical framework for observations previously 
dismissed as mere discrepancies!
This being Saturday, can the postulated time-dependent decay of previously 
observed observables also be applied to explain the apparent discrepancy in 
number of socks entering the wash vs. number recovered?
  Phoebe

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK 
] on behalf of Bernhard Rupp 
[hofkristall...@gmail.com ]
Sent: Friday, March 31, 2017 4:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Ligand electron density

Hi Fellows,
 
due to the change of month on Sa I am releasing already today
the first page of an until Sa embargoed reprint on concerns 
about theoretical principles of electron density reconstruction.
 
The paper is available on my dropbox (full paper after Sa): 
https://dl.dropboxusercontent.com/u/30662912/Rupp_2017_Phys_Rev_Letters_Electron_Density.pdf
 

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


Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] Twinning and R-Free

[benjamin bax]

Hi Alun,

1. What does phenix.xtriage think of the dataset?
2. What kind of redundancy do you have in the dataset?
3. Sometimes with a large crystal and a small beam the twin fraction varies as 
you rotate the crystal and illuminate different parts of the crystal (which can 
have different twin fractions). If you have enough data sometimes processing a 
subset of the frames will help you find a part of the crystal that has a 
consistent twin fraction.
4. I have had rod like p61 crystals where if you shoot either end of the rod 
they are not twinned, but if you shot the middle they looked twinned. They were 
just two crystals with opposite polarity growing together. You could have the 
same thing in I4. 

Ben

On 1 Mar 2017, at 22:03, Alun R Coker  wrote:

Hi Everyone,

I have a (2.5 - 2.3 Angstroms) data set the process as I4 or I222. Aimless 
indicates twinning in both space groups and all the lower symmetry space groups 
consistent with the reduced cell. Molecular replacement works in I4 but not 
I222 etc. I can see new density for a ligand we are interested in some of the 6 
subunits so all seems promising. I've refined in refmac without the twinning 
turned on in order to avoid model bias and end up with an R-factor/R-free of 
0.268/0.305. However, if I repeat the last round of refinement with twinning 
turned on the R-factor drops but not the R-free (R/R-free 0.237/0.305). Remac 
reckons the twin fraction is 0.628/0.372.

My question is does the drop in R-factor but not R-free suggest I am 
over-fitting the model by refining twinning?

Running the data and model through pdb redo suggest the data isn't twinned.

Thanks,

Alun

-- 
Dr Alun R. Coker
Senior Lecturer
Wolfson Institute for Biomedical Research
University College London
The Cruciform Building
London
WC1E 6BT

Tel: 020 7679 6703 Ext 46703
Web: www.ucl.ac.uk/pxmed


Dr Ben Bax

Dept. Biological Chemistry, 
John Innes Centre, 
Norwich Research Park, 
Norwich NR4 7UH, UK

ben.d.v@gmail.com

 

 

Re: [ccp4bb] Bad density for chains

[benjamin bax]

You could try the diffraction anisotropy server: 
http://services.mbi.ucla.edu/anisoscale/.
Sometimes it helps maps become more interpretable. 

 Ben  


On 26 Jan 2017, at 14:11, Pooja Kesari  wrote:

We have a 2.6 A structure showing four chains in an asymmetric unit. Our 
protein is 360 residues around 40 kDa . Mattews shows four chain in an 
assymetric unit (solvent 49% mattews coeff 2.44). The template has about 60% 
homologous with our protein. The molecular replacement against this template 
gave an initial free R of 38.  We did chain tracing and found that we have good 
density (2Fo-Fc) for chain A and B but poor density for C and D. 

1. The density for a particular stretch of 10 amino acids (disordered loop 
region) is absent in all the chains. We could not found density for this 
flexible loop region in any of the already known structures. Any suggestion on 
how can we build this region?

2. We did not find density for most of the loop regions in chain C and D which 
were well traced in chain A and B. How can we improving the density for these 
two chains based on chain A and B (Density modification)? 

3. We analysed the data using phenix xtriage and found that our data shows 
severe anisotropy. Any suggestion of anisotropy correction?

Pointless and Ctruncate analyses didn't show twinning or NCS.  I have checked 
the space group using Zanuda. We are stuck at a free value of 32. 

On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote:
This is a bit too vague to help much.
How did you solve the structure?
Eleanor

On 26 January 2017 at 03:50, Pooja Kesari mailto:pkesar...@gmail.com>> wrote:
Dear All,
Thank you all for reply.

We have checked the data for twinning.
Our protein is 360 residues around 40 kDa protein.
We have tried TLS refinement.
chain A and B don't superimpose well with chain C and D. (A and B chains also 
share slight difference )
Since we don't have proper density for some regions  chain C and D, we are not 
sure whether these chain have similar or different conformations. 
We tried anisotropy correction and the model refined a bit.


On Wed, Jan 25, 2017 at 10:32 AM, Debanu mailto:debanu@gmail.com>> wrote:
Hi Pooja,

Are you positive you have the correct space group and there are no other issues 
like twinning, etc?

If sure, did you define NCS groups in refinement? TLS refinement? Try different 
refinement programs?

How big is the molecule? Was it solved by MR or experimental phasing?

You can try superimposing A/B on C/D and refinement with tight NCS then adjust 
NCS restraints during model adjustments based on local differences or also see 
if phenix autobuild helps. 

Best,
Debanu 
--
Debanu Das
Accelero Biostructures 


On Jan 24, 2017, at 8:42 PM, Pooja Kesari mailto:pkesar...@gmail.com>> wrote:

> Dear All,
> 
> I have a 2.6 A resolution structure having four chains in an asymmetric unit.
> The chain A and B have density for almost all residues however we don't have 
> proper residue density in chain C and D.What can be tried to build chain C 
> and D ?
> 
> 
> 
> Many Thanks 
> Pooja



-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA





-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA



Dr Ben Bax

Dept. Biological Chemistry, 
John Innes Centre, 
Norwich Research Park, 
Norwich NR4 7UH, UK

ben.d.v@gmail.com