[ccp4bb] AW: [ccp4bb] MR solution not working

[Schreuder, Herman /DE]

Hi Shubhashish,
How many molecules do you assume are in the asymmetric unit? You may have a 
very high solvent content and by trying to find multiple molecules, you spoil 
your solution. Also, did you do a thermal shift assay to make sure your protein 
is properly folded?

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Shubhashish 
Chakraborty
Gesendet: Donnerstag, 3. März 2022 05:44
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] MR solution not working

Hello,
I am trying to solve a dataset using molecular replacement. However, neither 
Phaser MR nor Molrep can give any solution.
In Phaser, I have received an advisory that Top FTF has not packed.
I have tried molecular replacement using the wild-type protein at different 
resolutions (I am working on a mutant).
Also, I have truncated the loops from the input structure. However, none have 
worked.
So, what can be the possible way to solve this data set?

Thank you

Shubhashish Chakraborty
PhD JRF 2018
Structural and Molecular Biology Lab (Varma Lab)
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Khargar, Navi Mumbai
E-mail: schakrabo...@actrec.gov.in



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Re: [ccp4bb] MR solution not working

[Nicolas Foos]

Hello,

I have a maybe naive question, did you check the results for the Matthew 
Probabilities calculation ?


Are you 100% certain that's your protein of interest in the crystal? 
Sometimes we can have surprise.


Nicolas

On 03/03/2022 05:43, Shubhashish Chakraborty wrote:

Hello,
I am trying to solve a dataset using molecular replacement. However, 
neither Phaser MR nor Molrep can give any solution.

In Phaser, I have received an advisory that Top FTF has not packed.
I have tried molecular replacement using the wild-type protein at 
different resolutions (I am working on a mutant).
Also, I have truncated the loops from the input structure. However, 
none have worked.

So, what can be the possible way to solve this data set?
Thank you
Shubhashish Chakraborty
PhD JRF 2018
Structural and Molecular Biology Lab (Varma Lab)
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Khargar, Navi Mumbai
E-mail: schakrabo...@actrec.gov.in 



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--
Nicolas Foos PhD - ARISE fellow
https://orcid.org/-0003-2331-8399
   
EMBL Grenoble, McCarthy Team

71 av. des Martyrs,
38000 Grenoble FRANCE
   
+33 4 57 42 84 67




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Re: [ccp4bb] MR solution not working

[Mark J. van Raaij]

if the spacegroup is the same as wt and the cell params are similar, just do 
rigid body refinement and forget about MR (I’ll never understand people running 
MR needlessly in these cases…apart from wasting computer time it risks placing 
the new solution in a different place than the wt)
if the spacegroup is different, check cell parameters in the pdb and/or run 
Contaminer to see if you’ve crystallised a contaminant
try MR with packing restraints relaxed or switched off, just in case the mutant 
has loop in a new orientation that might clash with crystal neighbours in the 
old orientation.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 3 Mar 2022, at 05:43, Shubhashish Chakraborty 
> <750c9a1ca48b-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hello,
> I am trying to solve a dataset using molecular replacement. However, neither 
> Phaser MR nor Molrep can give any solution. 
> In Phaser, I have received an advisory that Top FTF has not packed. 
> I have tried molecular replacement using the wild-type protein at different 
> resolutions (I am working on a mutant).
> Also, I have truncated the loops from the input structure. However, none have 
> worked. 
> So, what can be the possible way to solve this data set?
>  
> Thank you
>  
> Shubhashish Chakraborty
> PhD JRF 2018
> Structural and Molecular Biology Lab (Varma Lab)
> Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
> Khargar, Navi Mumbai
> E-mail: schakrabo...@actrec.gov.in 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 
> 



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Re: [ccp4bb] MR solution not working

[Renato Weiße]

Dear Shubhashish,

in most cases the "just doing rigid body refinement" is working. But,  
depending on the space group, there are multiple possibilities for  
indexing the same set of reflections and then your simple refinement  
strategy will fail. Check the section "Alternative indexing" in the  
file  
https://www.ccp4.ac.uk/schools/DLS-2015/course_material/Datareduction2015.pdf  
for an example.


So, if the space group is indeed the same make sure that you use a  
dataset of one of the crystals of the native protein as a reference in  
the indexing step. It is also possible to provide it as reference in  
the scaling step with AIMLESS. POINTLESS is normally run before the  
actual scaling process and it will compare your data with the  
reference and will use the correct indexing scheme.


Cheers,
Renato


Zitat von "Mark J. van Raaij" :

if the spacegroup is the same as wt and the cell params are similar,  
just do rigid body refinement and forget about MR (I’ll never  
understand people running MR needlessly in these cases…apart from  
wasting computer time it risks placing the new solution in a  
different place than the wt)
if the spacegroup is different, check cell parameters in the pdb  
and/or run Contaminer to see if you’ve crystallised a contaminant
try MR with packing restraints relaxed or switched off, just in case  
the mutant has loop in a new orientation that might clash with  
crystal neighbours in the old orientation.



Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


On 3 Mar 2022, at 05:43, Shubhashish Chakraborty  
<750c9a1ca48b-dmarc-requ...@jiscmail.ac.uk> wrote:


Hello,
I am trying to solve a dataset using molecular replacement.  
However, neither Phaser MR nor Molrep can give any solution.

In Phaser, I have received an advisory that Top FTF has not packed.
I have tried molecular replacement using the wild-type protein at  
different resolutions (I am working on a mutant).
Also, I have truncated the loops from the input structure. However,  
none have worked.

So, what can be the possible way to solve this data set?

Thank you

Shubhashish Chakraborty
PhD JRF 2018
Structural and Molecular Biology Lab (Varma Lab)
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Khargar, Navi Mumbai
E-mail: schakrabo...@actrec.gov.in 
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1  





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--
Dr. Renato Weiße
Biotechnologisch-Biomedizinisches Zentrum
Institut für Bioanalytische Chemie
Strukturanalytik von Biopolymeren
Universität Leipzig
Deutscher Platz 5
04103 Leipzig

Mail  renato.wei...@bbz.uni-leipzig.de
Tel.  +49 341 97 31316
Fax   +49 341 97 31131310



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[ccp4bb] Structural Biologist Wanted at the Franklin

[James H Naismith]

https://opportunities.rfi.ac.uk/vacancy/postdoctoral-research-scientist-in-protein-science-477711.html
 

 
Dear All,
Just a note to say Ray Owens and I have a vacancy to work on the structural 
biology of nanobodies.
The Franklin is a new research institute, it is based on the Harwell Campus 
next door to Diamond.
Our new building is a fabulous place to work.

The star of the our nanobody team is Fifi The Franklin Llama, a regular on TV 
shows including the Christmas Lectures.

Surely the chance to work with such a media celebrity is enticement enough.

If you need more to convince you, look at the website or read our papers.

Ray (ray.ow...@strubi.ox.ac.uk ) or I happy 
to chat informally.


With best wishes
Jim

James H Naismith FRS FRSE FMedSci
Director of the Rosalind Franklin Institute, Harwell Campus, OX11 0QS
Phone: 01235 567701


The Rosalind Franklin Institute is a registered charity in England and Wales, 
No. 1179810
Company Limited by Guarantee Registered in England and Wales, No. 11266143
Funded by UK Research and Innovation through the Engineering and Physical 
Sciences Research Council

ResearcherID H-3408-2012; SCOPUS_ID 7005691850;  ORCID -0001-6744-5061

I am ResearchGate as James_Naismith
I am on linkedin as Jim Naismith

Pronouns: he, him, his 




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Re: [ccp4bb] Ligand occupancy refinement

[Mahmoud RIZK]

Hello,


What about refining using refmac5 ?

it may yield to different results



Mahmoud Rizk


On 03/03/2022 15:15, Akanksha Tomar wrote:

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing 
the protein model and water I fitted the ligand into it. Currently, I 
am using Phenix Refine with occupancy refinement for individual atoms 
switched on. After the refinement, the overall occupancy of the ligand 
is 0.7 and the RSCC value is 0.86. The resolution of the structure is 
2.1 Å.


Now the problem is that the program has assigned different occupancies 
to different atoms of the ligand. For some cases, it has assigned 0 
occupancies to atoms for which there is a clear positive peak.


Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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[ccp4bb] Ligand occupancy refinement

[Akanksha Tomar]

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing the
protein model and water I fitted the ligand into it. Currently, I am using
Phenix Refine with occupancy refinement for individual atoms switched on.
After the refinement, the overall occupancy of the ligand is 0.7 and the
RSCC value is 0.86. The resolution of the structure is 2.1 Å.

Now the problem is that the program has assigned different occupancies to
different atoms of the ligand. For some cases, it has assigned 0
occupancies to atoms for which there is a clear positive peak.

Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

-- 
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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Re: [ccp4bb] Ligand occupancy refinement

[Wim Burmeister]

Hello, 
at 2.1 A resolution, atomic temperature factors and occupancy are strongly 
correlated. So you have to be very careful with the results. 
So the best is just to set the inhibitor to the average occupancy and then to 
include it into a full positional and B-factor refinement. You can check 
whether the result is coherent by comparing the B-factors of the ligand and of 
the atoms, which are in contact with it. If this is not the case, you may want 
to adjust the occupancy manually. As there are also solvent atoms at the ligand 
positions, when it is not bound, there is another source of inaccuracy and 
theoretically you would have to model the site with the solvent and an 
occupancy 1-q and the ligand with an occupancy q as alternate structures. But 
nobody does that and it is not really required. 
Best 
Wim 


De: "Akanksha Tomar"  
À: "CCP4BB"  
Envoyé: Jeudi 3 Mars 2022 15:15:07 
Objet: [ccp4bb] Ligand occupancy refinement 

Hi everyone, 

I am trying to refine the occupancy of a bound ligand. After fixing the protein 
model and water I fitted the ligand into it. Currently, I am using Phenix 
Refine with occupancy refinement for individual atoms switched on. After the 
refinement, the overall occupancy of the ligand is 0.7 and the RSCC value is 
0.86. The resolution of the structure is 2.1 Å. 

Now the problem is that the program has assigned different occupancies to 
different atoms of the ligand. For some cases, it has assigned 0 occupancies to 
atoms for which there is a clear positive peak. 

Why it has been done so and is it acceptable? 

Any help would be greatly appreciated. 

Thank you. 

-- 
Best Regards, 
Akanksha Tomar 
Pre-Doctoral Fellow, 
C\o Dr. Arockiasamy Arulandu, 
Membrane Protein Biology Group, 
International Center for Genetic Engineering and Biotechnology, 
New Delhi, India 




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-- 
Wim Burmeister 
Professor 
Institut de Biologie Structurale (IBS) CIBB 
71 avenue des Martyrs / CS 20192 
38044 Grenoble Cedex 9, FRANCE 
E-mail: [ mailto:wim.burmeis...@ibs.fr | wim.burmeis...@ibs.fr ] 
Mobile: +33 (0) 7 50 49 19 91 
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Re: [ccp4bb] Ligand occupancy refinement

[Jon Cooper]

Hello, the ligand needs to be treated as one occupancy group since refining 
individual occupancies would be a case of refining too many parameters, unless 
it was a very fragmentary compound!! It is one keyword in refmac, but I can't 
remember for phenix, sorry! Ta jc

Sent from ProtonMail mobile

 Original Message 
On 3 Mar 2022, 14:15, Akanksha Tomar wrote:

> Hi everyone,
>
> I am trying to refine the occupancy of a bound ligand. After fixing the 
> protein model and water I fitted the ligand into it. Currently, I am using 
> Phenix Refine with occupancy refinement for individual atoms switched on. 
> After the refinement, the overall occupancy of the ligand is 0.7 and the RSCC 
> value is 0.86. The resolution of the structure is 2.1 Å.
>
> Now the problem is that the program has assigned different occupancies to 
> different atoms of the ligand. For some cases, it has assigned 0 occupancies 
> to atoms for which there is a clear positive peak.
>
> Why it has been done so and is it acceptable?
>
> Any help would be greatly appreciated.
>
> Thank you.
>
> --
>
> Best Regards,
> Akanksha Tomar
> Pre-Doctoral Fellow,
> C\o Dr. Arockiasamy Arulandu,
> Membrane Protein Biology Group,
> International Center for Genetic Engineering and Biotechnology,
> New Delhi, India
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



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Re: [ccp4bb] Ligand occupancy refinement

[Weiergräber , Oliver H .]

Dear Akanksha Tomar,

By default, phenix.refine will assign a single occupancy for the ligand as long 
as all atoms have the _same_ occupancy (0https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1





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[ccp4bb] AW: [ccp4bb] Ligand occupancy refinement

[Schreuder, Herman /DE]

Dear Ankanksha,

The ligand is either present, or it is not present. It cannot be that some 
atoms are present and others not. For ligands, I always use a single group 
occupancy using the program Buster from global phasing. In my hands, this 
always works. There is a correlation between occupancy and B-factors, but if 
you use a single occupancy for a large number of atoms, this correlation is not 
longer a problem. Also, modern maximum likelihood refinement programs do a good 
job in separating the contributions of occupancy and B-factors.

Best,
Herman

Von: CCP4 bulletin board  Im Auftrag von Jon Cooper
Gesendet: Donnerstag, 3. März 2022 16:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Ligand occupancy refinement

Hello, the ligand needs to be treated as one occupancy group since refining 
individual occupancies would be a case of refining too many parameters, unless 
it was a very fragmentary compound!! It is one keyword in refmac, but I can't 
remember for phenix, sorry! Ta jc


Sent from ProtonMail mobile



 Original Message 
On 3 Mar 2022, 14:15, Akanksha Tomar < 
akankshat...@gmail.com> wrote:

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing the protein 
model and water I fitted the ligand into it. Currently, I am using Phenix 
Refine with occupancy refinement for individual atoms switched on. After the 
refinement, the overall occupancy of the ligand is 0.7 and the RSCC value is 
0.86. The resolution of the structure is 2.1 Å.

Now the problem is that the program has assigned different occupancies to 
different atoms of the ligand. For some cases, it has assigned 0 occupancies to 
atoms for which there is a clear positive peak.

Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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[ccp4bb] AW: [ccp4bb] Ligand occupancy refinement

[Schreuder, Herman /DE]

PS: this does not work for very small atoms, e.g. waters. Here I let the 
temperature factor take care of the occupancy as well.

Von: Schreuder, Herman /DE
Gesendet: Donnerstag, 3. März 2022 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: AW: [ccp4bb] Ligand occupancy refinement

Dear Ankanksha,

The ligand is either present, or it is not present. It cannot be that some 
atoms are present and others not. For ligands, I always use a single group 
occupancy using the program Buster from global phasing. In my hands, this 
always works. There is a correlation between occupancy and B-factors, but if 
you use a single occupancy for a large number of atoms, this correlation is not 
longer a problem. Also, modern maximum likelihood refinement programs do a good 
job in separating the contributions of occupancy and B-factors.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Jon Cooper
Gesendet: Donnerstag, 3. März 2022 16:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Ligand occupancy refinement

Hello, the ligand needs to be treated as one occupancy group since refining 
individual occupancies would be a case of refining too many parameters, unless 
it was a very fragmentary compound!! It is one keyword in refmac, but I can't 
remember for phenix, sorry! Ta jc


Sent from ProtonMail mobile



 Original Message 
On 3 Mar 2022, 14:15, Akanksha Tomar < 
akankshat...@gmail.com> wrote:

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing the protein 
model and water I fitted the ligand into it. Currently, I am using Phenix 
Refine with occupancy refinement for individual atoms switched on. After the 
refinement, the overall occupancy of the ligand is 0.7 and the RSCC value is 
0.86. The resolution of the structure is 2.1 Å.

Now the problem is that the program has assigned different occupancies to 
different atoms of the ligand. For some cases, it has assigned 0 occupancies to 
atoms for which there is a clear positive peak.

Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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Re: [ccp4bb] Ligand occupancy refinement

[Akanksha Tomar]

Thank you for the suggestions
I will try again by setting the occupancy of the entire ligand to the
single average occupancy and re-do the refinement with Buster, Phenix
refine and Remac5 with full positional and B-factor refinement and check
the B-factor of the neighbouring residues.
The ligand is a 30 atom containing molecule binding at a shallow
solvent-exposed site.



On Thu, 3 Mar 2022 at 20:38, Wim Burmeister  wrote:

> Hello,
> at 2.1 A resolution, atomic temperature factors and occupancy are strongly
> correlated. So you have to be very careful with the results.
> So the best is just to set the inhibitor to the average occupancy and then
> to include it into a full positional and B-factor refinement. You can check
> whether the result is coherent by comparing the B-factors of the ligand and
> of the atoms, which are in contact with it. If this is not the case, you
> may want to adjust the occupancy manually. As ther are also solvent atoms
> at the ligand positions, when it is not bound, there is another source of
> inaccuracy and theoretically you would have to model the site with the
> solvent and an occupancy 1-q and the ligand with an occupancy q as
> alternate structures. But nobody does that and it is not really required.
> Best
> Wim
>
> --
> *De: *"Akanksha Tomar" 
> *À: *"CCP4BB" 
> *Envoyé: *Jeudi 3 Mars 2022 15:15:07
> *Objet: *[ccp4bb] Ligand occupancy refinement
>
> Hi everyone,
>
> I am trying to refine the occupancy of a bound ligand. After fixing the
> protein model and water I fitted the ligand into it. Currently, I am using
> Phenix Refine with occupancy refinement for individual atoms switched on.
> After the refinement, the overall occupancy of the ligand is 0.7 and the
> RSCC value is 0.86. The resolution of the structure is 2.1 Å.
>
> Now the problem is that the program has assigned different occupancies to
> different atoms of the ligand. For some cases, it has assigned 0
> occupancies to atoms for which there is a clear positive peak.
>
> Why it has been done so and is it acceptable?
>
> Any help would be greatly appreciated.
>
> Thank you.
>
> --
> Best Regards,
> Akanksha Tomar
> Pre-Doctoral Fellow,
> C\o Dr. Arockiasamy Arulandu,
> Membrane Protein Biology Group,
> International Center for Genetic Engineering and Biotechnology,
> New Delhi, India
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
>
> --
> *Wim Burmeister*
> Professor
> Institut de Biologie Structurale (IBS) CIBB
> 71 avenue des Martyrs
> 
> / CS 20192
> 38044 Grenoble Cedex 9, FRANCE
> E-mail: wim.burmeis...@ibs.fr
> Mobile:   +33 (0) 7 50 49 19 91
> website
> 
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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Re: [ccp4bb] Why there are two solutions about heavy atoms in SHELXD

[Kay Diederichs]

On Wed, 2 Mar 2022 22:56:28 +0800, fu  wrote:

>Dear Colleagues,
>   I get two solutions after running SHELX C/D. In both results, the 
> coordinates of the heavy atoms are centrosymmetric. Why there are two 
> solutions?
>
>Best wishes,
>Fu Xingke
>Institute of Physics CAS

Hello Fu,

SHELXD writes a single .res and a single .pdb file. So how do you get two 
solutions?

Just a hint about asking questions on CCP4BB: you should include more precise 
information. Then you get better answers.
- which operating system, which program, which version of program, which 
version of CCP4 (may not be very relevant for this specific problem, but 
generally it is helpful to know)
- what is the resolution of the data? Which data reduction program?
- what is the input to SHELXD? Show the .ins file !
- what is the output of SHELXD? Show the .res file !

best,
Kay



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Re: [ccp4bb] Why there are two solutions about heavy atoms in SHELXD

[Kay Diederichs]

On Wed, 2 Mar 2022 22:56:28 +0800, fu  wrote:

>Dear Colleagues,
>   I get two solutions after running SHELX C/D. In both results, the 
> coordinates of the heavy atoms are centrosymmetric. Why there are two 
> solutions?
>
>Best wishes,
>Fu Xingke
>Institute of Physics CAS

Hello Fu,

SHELXD writes a single .res and a single .pdb file. So how do you get two 
solutions?

Just a hint about asking questions on CCP4BB:  more precise information enables 
better answers.
- which operating system, which program, which version of program, which 
version of CCP4 (may not be very relevant for this specific problem, but 
generally helpful to know)
- what is the resolution of the data? Which data reduction program? (may not be 
very relevant for this specific problem, but often helpful to know)
- what is the input to SHELXD? Show the .ins file !
- what is the output of SHELXD? Show the .res file !

best,
Kay



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

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Re: [ccp4bb] Ligand occupancy refinement

[mesters]

Hi Akanksha,

years ago I tried to refine, using refmac, an inhibitor only with 
partial occupancy bound to HIV-1 PR and was not that happy (probably a 
biased view) with the outcome.
In a next step I included water molecules with partial occupancies that 
normally occupy the binding site in the absence of inhibitor but was not 
happy at all because especially the water molecules, despite partial 
occupancies and total occupancy of both water and inhibitor equal to 1, 
shifted to positions deviating from the positions observed in the 
uninhibited structure. For a refinement involving the inhibitor only I 
can understand some drift of the inhibitor which is related to the 
position/density of the partial waters that were not included in the 
model..


In a next step I then compared NCS, Refmac and SHELXL refinement with 
partial occupancy for inhibitor + waters in the binding site and spend 
weeks optimizing the parameters in the different programs.


End of story, SHELX - based on a visual inspection of the final ED maps 
- came closest to what I considered acceptable but again, maybe a biased 
view (did not test Buster tough!):


What I learned from this "partial occupancy" odyssee:
1) Different programs handled this type of "constellation" differently
2) Including partial waters did make a difference altough it was/is not 
common practice
3) Inhibitor occupancy and position drifted somewhat comparing with or 
without waters


As already pointed out, you can not have different occupancies for the 
different atoms of the ligand. At 2.1 Å resolution, test several fixed 
occupancies - start at 0.5 and increase by 0.1 - and compare/inspect the 
different ED maps along witht the R/Rfree etc. and pick the occupancy 
that makes most sense


Good luck,

Jeroen




Am 03.03.22 um 16:08 schrieb Wim Burmeister:

Hello,
at 2.1 A resolution, atomic temperature factors and occupancy are 
strongly correlated. So you have to be very careful with the results.
So the best is just to set the inhibitor to the average occupancy and 
then to include it into a full positional and B-factor refinement. You 
can check whether the result is coherent by comparing the B-factors of 
the ligand and of the atoms, which are in contact with it. If this is 
not the case, you may want to adjust the occupancy manually. As there 
are also solvent atoms at the ligand positions, when it is not bound, 
there is another source of inaccuracy and theoretically you would have 
to model the site with the solvent and an occupancy 1-q and the ligand 
with an occupancy q as alternate structures. But nobody does that and 
it is not really required.

Best
Wim


*De: *"Akanksha Tomar" 
*À: *"CCP4BB" 
*Envoyé: *Jeudi 3 Mars 2022 15:15:07
*Objet: *[ccp4bb] Ligand occupancy refinement

Hi everyone,

I am trying to refine the occupancy of a bound ligand. After fixing 
the protein model and water I fitted the ligand into it. Currently, I 
am using Phenix Refine with occupancy refinement for individual atoms 
switched on. After the refinement, the overall occupancy of the ligand 
is 0.7 and the RSCC value is 0.86. The resolution of the structure is 
2.1 Å.


Now the problem is that the program has assigned different occupancies 
to different atoms of the ligand. For some cases, it has assigned 0 
occupancies to atoms for which there is a clear positive peak.


Why it has been done so and is it acceptable?

Any help would be greatly appreciated.

Thank you.

--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India



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--
*Wim Burmeister*
Professor
Institut de Biologie Structurale (IBS) CIBB
71 avenue des Martyrs / CS 20192
38044 Grenoble Cedex 9, FRANCE
E-mail: wim.burmeis...@ibs.fr
Mobile:   +33 (0) 7 50 49 19 91
website 
 






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--
signature.html *Dr. /math. et dis. nat./ Jeroen R. Mesters
*Deputy, Lecturer, Program Coordinator Infection Biology 

Visiting Professorship (South Bohemian University 
) in Biophysics


*University of Lübeck*
Cent

[ccp4bb] Online Workshop Series on Structural and Crystallographic Databases -- Registration Open

[Mitchell D. Miller]

On behalf of The U.S. National Committee for Crystallography (USNC/Cr)  
of the National Academies of Sciences, Engineering, and Medicine, I am  
happy to announce that registration is now open for "EXPLORING  
STRUCTURAL DATABASE USE IN CRYSTALLOGRAPHY: WORKSHOP SERIES".  The  
topics will focus on using, developing, and maintaining  
crystallographic and structural databases- encompassing  
macromolecular, small molecule, and powder diffraction.


We will open the series with a keynote delivered by John Helliwell  
(IUCr Chair of the Committee on Data) on March 21, 2022 followed by 10  
one-day and two-day workshops focused on 8 different structural and  
crystallographic data resources. An overview of the program can be  
found in the attached PDF.


Graduate students, postdoctoral fellows, faculty members and  
researchers in any of the crystallographic, diffraction, and imaging  
sciences affiliated with the International Union of Crystallography  
(IUCr) are encouraged to register and participate in the training  
sessions that interest them.


Registration for this online series is free. To read more about each  
session and to register, please visit:

https://www.nationalacademies.org/our-work/exploring-structural-database-use-in-crystallography-a-usnccr-workshop-series
The details about each session can be found by clicking the event  
pages along with a registration link allows one to register for any  
combination of the 1 and 2 day sessions (each meeting for 2-4 hrs/day).


The workshop series is sponsored by the National Institute of  
Standards and Technology (NIST).


For more information, please contact Ana Ferreras, Telephone: (202)  
334-1697 Email: aferre...@nas.edu[1]


Sincerely,
Mitch


Links:
--
[1]



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Crystallographic-Database-Workshop-Flyer-v2.pdf
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