Re: [ccp4bb] high B factor

[Daniel M. Himmel, Ph. D.]

By the way, I would recommend running simulated annealing with each
composite omit map you generate to help in overcoming model bias.

-Daniel


On Sat, Nov 10, 2018 at 9:31 PM Daniel M. Himmel, Ph. D. <
danielmhim...@gmail.com> wrote:

> Anandhi,
>
> Assuming the data reduction went well, and you're in the right space goup,
> there could be a lot of model bias in your structure stemming from the
> starting model.
>
> There are a lot of things to try.  I would
> set all the B-factors to an artificially low B-factor to help de-mask
> errors.  Then,
> you can generate a composite omit map and FEM maps to see if any obvious
> model errors show up in the electron density.  After correcting these, you
> can try running
> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
> each of these, especially in regions where your original model had high
> b-factors.
> Use Coot to identify areas of poor agreement with electron density and
> areas
> of poor geometry.  Once you spent a while correcting the model, put it
> through
> one or more cycles of simulated annealing at different temperatures in
> parallel.
> Select several sets of coordinates that give the best Rfree convergence,
> and then
> subject those models to individual B-factor refinement.  After that, check
> again in Coot
> for areas of high B-factors and areas of poor geometry (try especially to
> improve
> your Ramachandran Plot).  Use MolProbity to help in identifying errors and
> clashes.
> If a few rounds of simulated annealing and model building don't improve
> things, try some
> refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help
> give you alternative models.  While doing all these, don't be afraid to
> "cut-and-paste"
> regions from one model into another model and then correct the geometry in
> Coot.
> If B-factors don't come down no matter what you do, you could be in the
> wrong space
> group or have problems with the original data that need to be addressed.
>
> I hope this helps.
>
> Daniel
>
> ___
>
> Daniel M. Himmel, Ph. D.
>
> URL:  http://www.DanielMHimmel.com/index_Xtal.html
> 
>
>
>
>
> On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan 
> wrote:
>
>> Hello everyone,
>>
>>
>> I am trying to solve the structure of a protein with a bound ligand at
>> 2.65 A resolution. XDS was used for data reduction, phaser-MR for molecular
>> replacement  and Phenix for refinement. The refinement was done with the
>> default settings ( individual B factors, occupancy and TLS parameters). The
>> resultant atomic B factors are quite high.  The overall B factor is 82 with
>> a minimum value of 34.57 and maximum of 225.13. I would like to know if any
>> of the data reduction parameters can affect the B factors and how best to
>> deal with this issue.
>>
>>
>> Anandhi
>>
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>



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Re: [ccp4bb] high B factor

[Daniel M. Himmel, Ph. D.]

Anandhi,

Assuming the data reduction went well, and you're in the right space goup,
there could be a lot of model bias in your structure stemming from the
starting model.

There are a lot of things to try.  I would
set all the B-factors to an artificially low B-factor to help de-mask
errors.  Then,
you can generate a composite omit map and FEM maps to see if any obvious
model errors show up in the electron density.  After correcting these, you
can try running
Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
each of these, especially in regions where your original model had high
b-factors.
Use Coot to identify areas of poor agreement with electron density and areas
of poor geometry.  Once you spent a while correcting the model, put it
through
one or more cycles of simulated annealing at different temperatures in
parallel.
Select several sets of coordinates that give the best Rfree convergence,
and then
subject those models to individual B-factor refinement.  After that, check
again in Coot
for areas of high B-factors and areas of poor geometry (try especially to
improve
your Ramachandran Plot).  Use MolProbity to help in identifying errors and
clashes.
If a few rounds of simulated annealing and model building don't improve
things, try some
refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help
give you alternative models.  While doing all these, don't be afraid to
"cut-and-paste"
regions from one model into another model and then correct the geometry in
Coot.
If B-factors don't come down no matter what you do, you could be in the
wrong space
group or have problems with the original data that need to be addressed.

I hope this helps.

Daniel

___

Daniel M. Himmel, Ph. D.

URL:  http://www.DanielMHimmel.com/index_Xtal.html





On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan 
wrote:

> Hello everyone,
>
>
> I am trying to solve the structure of a protein with a bound ligand at
> 2.65 A resolution. XDS was used for data reduction, phaser-MR for molecular
> replacement  and Phenix for refinement. The refinement was done with the
> default settings ( individual B factors, occupancy and TLS parameters). The
> resultant atomic B factors are quite high.  The overall B factor is 82 with
> a minimum value of 34.57 and maximum of 225.13. I would like to know if any
> of the data reduction parameters can affect the B factors and how best to
> deal with this issue.
>
>
> Anandhi
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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[ccp4bb] Clashes in Phaser

[D Bonsor]

I am currently trying to solve two datasets of two different protein-protein 
complexes. One of the proteins is common to both complexes and there is a 
deposited structure in the PDB (100% identical). The other proteins currently 
have not been solved and share ~50% identity between them. Both complexes 
crystallize in different space groups. All three proteins are ~12kDa. Both 
datasets diffract to ~3-3.5A.

A) C2221 109.6 110.1 335.2 90 90 90 with around 9 copies of the complex.
B) I4122 131.6 131.6 257.1 90 90 90 with 5 copies of the complex.

I am slightly suspicious of Dataset A due to similar a and b lengths. Merging 
in P4, results in higher Rmerges. 

However in each case, searching with the common protein, results in Phaser 
failing to find a single copy of the protein, despite TFZ scores of 8-12, due 
to rejection during packing;

Dataset A
   Packing Table
   -
   Solutions accepted if total number of clashes <= 5% of trace atoms
  i.e. total number of clashes <= 5
   AND if number of clashes <= 5% of trace atoms for each ensemble
  i.e. ensemble1: number of clashes <= 5
   ##  #Clash Packs SpaceGroup  Annotation
   1 198   NO   C 2 2 2 RFZ=2.9 TFZ=11.2
   2 154   NO   C 2 2 2 RFZ=2.3 TFZ=10.8
   3 229   NO   C 2 2 2 RFZ=2.6 TFZ=11.5
   4 161   NO   C 2 2 2 RFZ=2.5 TFZ=10.0
   5 118   NO   C 2 2 2 RFZ=2.4 TFZ=9.8
   6 166   NO   C 2 2 2 RFZ=2.9 TFZ=8.1
   7 199   NO   C 2 2 2 RFZ=2.6 TFZ=10.0
   8 165   NO   C 2 2 2 RFZ=2.5 TFZ=10.0
   9 167   NO   C 2 2 2 RFZ=2.4 TFZ=10.1
   10168   NO   C 2 2 2 RFZ=2.5 TFZ=8.8
   11186   NO   C 2 2 2 RFZ=2.4 TFZ=9.0
   1293NO   C 2 2 2 RFZ=2.5 TFZ=8.3
   13155   NO   C 2 2 2 RFZ=3.1 TFZ=8.8
   14149   NO   C 2 2 2 RFZ=2.9 TFZ=8.1
   15158   NO   C 2 2 2 RFZ=2.6 TFZ=9.7
   16151   NO   C 2 2 2 RFZ=2.6 TFZ=10.5
   17147   NO   C 2 2 2 RFZ=3.1 TFZ=8.6
   18145   NO   C 2 2 2 RFZ=2.4 TFZ=8.8
   19130   NO   C 2 2 2 RFZ=2.4 TFZ=10.8
   20166   NO   C 2 2 2 RFZ=2.9 TFZ=8.4
   21215   NO   C 2 2 2 RFZ=2.4 TFZ=10.0
   22147   NO   C 2 2 2 RFZ=2.6 TFZ=10.1
   23189   NO   C 2 2 2 RFZ=2.6 TFZ=9.6
   24164   NO   C 2 2 2 RFZ=2.5 TFZ=9.2
   25166   NO   C 2 2 2 RFZ=2.6 TFZ=9.3
   26234   NO   C 2 2 2 RFZ=2.4 TFZ=8.3
   27170   NO   C 2 2 2 RFZ=2.4 TFZ=9.3
   28126   NO   C 2 2 2 RFZ=2.6 TFZ=8.1
   29189   NO   C 2 2 2 RFZ=2.5 TFZ=9.4
   30171   NO   C 2 2 2 RFZ=2.3 TFZ=9.6
   31211   NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   32203   NO   C 2 2 2 RFZ=2.5 TFZ=10.2
   33162   NO   C 2 2 2 RFZ=2.4 TFZ=9.7
   34116   NO   C 2 2 2 RFZ=2.6 TFZ=8.5
   35168   NO   C 2 2 2 RFZ=2.5 TFZ=8.1
   36159   NO   C 2 2 2 RFZ=2.4 TFZ=9.6
   3791NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   38114   NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   39212   NO   C 2 2 2 RFZ=2.4 TFZ=8.3
   40162   NO   C 2 2 2 RFZ=2.4 TFZ=10.1
   41182   NO   C 2 2 2 RFZ=2.9 TFZ=8.4
   42112   NO   C 2 2 2 RFZ=2.5 TFZ=8.3
   43131   NO   C 2 2 2 RFZ=2.4 TFZ=9.3
   44166   NO   C 2 2 2 RFZ=2.3 TFZ=9.4
   45135   NO   C 2 2 2 RFZ=2.4 TFZ=9.4
   46156   NO   C 2 2 2 RFZ=2.5 TFZ=8.5
   47189   NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   48133   NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   49200   NO   C 2 2 2 RFZ=2.5 TFZ=9.1
   50146   NO   C 2 2 2 RFZ=2.6 TFZ=9.2
   51117   NO   C 2 2 2 RFZ=2.4 TFZ=8.5
   52128   NO   C 2 2 2 RFZ=2.5 TFZ=8.2
   53144   NO   C 2 2 2 RFZ=2.4 TFZ=9.0
   54148   NO   C 2 2 2 RFZ=2.5 TFZ=8.1
   55119   NO   C 2 2 2 RFZ=2.4 TFZ=8.5
   56131   NO   C 2 2 2 RFZ=2.4 TFZ=8.5
   57131   NO   C 2 2 2 RFZ=2.5 TFZ=8.4
   58173   NO   C 2 2 2 RFZ=2.6 TFZ=8.9
   59114   NO   C 2 2 2 RFZ=2.6 TFZ=8.1
   60150   NO   C 2 2 2 RFZ=2.4 TFZ=8.6
   61150   NO   C 2 2 2 RFZ=2.4 TFZ=8.4
   62138   NO   C 2 2 2 RFZ=2.3 TFZ=8.6
   63123   NO   C 2 2 2 RFZ=2.5 TFZ=8.3
   64122   NO   C 2 2 2 RFZ=2.5 TFZ=8.2
   65116   NO   C 2 2 2 RFZ=2.6 TFZ=8.4
   66118   NO   C 2 2 2 RFZ=2.4 TFZ=8.0
   67131   NO   C 2 2 2 RFZ=2.4 TFZ=8.3
   6896NO   C 2 2 2 RFZ=2.4 TFZ=8.4
   6998NO   C 2 2 2 RFZ=2.4 TFZ=8.0
   70198   NO   C 2 2 2 RFZ=2.5 TFZ=8.6
   71137   NO   C 2 2 2 RFZ=2.4 TFZ=8.9
   72110   NO   C 2 2 2 RFZ=2.5 TFZ=8.2
   7370NO   C 2 2 2 RFZ=2.4 TFZ=8.4
   74128   NO  

Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

[Tomas Malinauskas]

Dear Michael,

On Sat, Nov 10, 2018 at 9:56 AM Michael Jarva  wrote:
>
> Dear ccp4 community,
>
>
> I have recently been working with a structure that has an Asparagine that 
> makes a planar stacking connection with a Tryptophan ring 
> (pep_ASN-TRP_v2.png), that seem to be a true pi-stacking interaction and I'd 
> like to find more examples of this.

You could check the following paper:
https://elifesciences.org/articles/31486
Pi-Pi contacts are an overlooked protein feature relevant to phase separation

They have a list of examples (including Asn-Trp pi-pi stacking) in PDB
(Figure 1, data source):
Pi-Pi contact annotations for the full PDB set.
Text file listing the pi-pi contacts observed across our non-redundant
PDB set, with contact types shown by residue annotations where single
amino acid names refer to sidechains and pairs of amino acids refer to
the backbone peptide bond between residue i and residue i + 1.
https://doi.org/10.7554/eLife.31486.005

Best wishes,
Tomas

Dr. Tomas Malinauskas
University of Oxford
Wellcome Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford OX3 7BN
United Kingdom
to...@strubi.ox.ac.uk
tomas.malinaus...@gmail.com



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Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

[William G. Scott]

Hi Michael:

It always makes me happy to see that there are people who care about this.

3.3 to 3.4 Å should be an ideal distance for this, and, as you note, the lone 
pair
residing on the (sp^3-hybridized) nitrogen would have to be oriented for 
favorable overlap, which is a bit harder to deduce from your figure.

The other rotomer would place oxygen at that position. Because it is 
double-bonded
to the gamma carbon, the lone pairs are oriented differently, and the pi-bond 
would
be approximately parallel to the plane containing the tryptophan, which would 
give
a nice pi-stacking interaction similar to what is seen with adjacent base pairs 
in nucleic acids.

Expectation bias, as well as having partial charges turned on during 
refinement, might
influence the rotomeric state of examples from the PDB, so be careful of social 
consensus.
With classical electostatics, N has a partial positive charge, and O has a 
partial negative 
charge.  (If you think about it, all pi-stacking interactions, from the point 
of view of classical
electrostatics, would be somewhat repulsive.)

Good luck with this, and let us know of the outcome.


Bill



William G. Scott
Director, Program in Biochemistry and Molecular Biology
Professor, Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
University of California at Santa Cruz
Santa Cruz, California 95064
USA

http://scottlab.ucsc.edu

> On Nov 10, 2018, at 1:45 AM, Michael Jarva  wrote:
> 
> Dear ccp4 community,
> 
> I have recently been working with a structure that has an Asparagine that 
> makes a planar stacking connection with a Tryptophan ring 
> (pep_ASN-TRP_v2.png), that seem to be a true pi-stacking interaction and I'd 
> like to find more examples of this.
> 
> I've found a few other examples in the literature but they are mostly amide 
> hydrogen-pi interactions (4PTI_ASN-TYR_v2.png and 1N4W_ASN-FAD_v2.png), which 
> can be seen by the way the Asn is dipping down into the pi-cloud. One 
> potential exception is of a DNA-binding protein where the orientation is more 
> planar (3HXQ_GLN-DNA_v2.png).
> 
> So, I'm looking for examples of Asparagine or Glutamine pi-stacking and am 
> sure there are more of them out there and would greatly appreciate any 
> examples.
> 
> best regards
> Michael
> 
> Michael Jarva, PhD
> ACRF Chemical Biology Division
> The Walter and Eliza Hall Institute of Medical Research
> 1G Royal Parade
> Parkville Victoria 3052
> Australia
> Phone: +61 3 9345 2493 
> Email: jarv...@wehi.edu.au | Web: http://www.wehi.edu.au/
> The ACRF Chemical Biology Division is supported by the
> Australian Cancer Research Foundation
> 
> ___ 
> 
> The information in this email is confidential and intended solely for the 
> addressee.
> You must not disclose, forward, print or use it without the permission of the 
> sender.
> 
> The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
> Kulin 
> Nation as the traditional owners of the land where our campuses are located 
> and 
> the continuing connection to country and community.
> ___
> 
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> <4PTI_ASN-TYR_v2.png><3HXQ_GLN-DNA_v2.png><1N4W_ASN-FAD_v2.png>



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