A few questions

Rife, Christopher L

6 Feb 2008 6 Feb '08

4:27 p.m.

Hi, We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx. We followed the guidelines recently posted to this BB, and initially the results looked pretty good, but upon close inspection we have a few questions. We are using Version: 2008_01_09_2058. 1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual? 2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O 3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens. I can make the files available if that will help. Thanks, Chris ----------------------------------------- Structure Determination Core Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory

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Pavel Afonine

6 Feb 6 Feb

4:56 p.m.

Hi Christopher, thanks for your questions!

...

We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...

1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that this is not "usual amino acid, water, dna/rna fragment".

...

2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C the total occupancy is: 0.77+0.23=1 Same for other atoms.

...

3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis. Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this: hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual } Please let us know if you have any questions or problems! Pavel.

Paul Adams

4:59 p.m.

Hi Pavel, the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies. Cheers, Paul On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...

Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that this is not "usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

Peter Zwart

5:02 p.m.

I guess what is needed is not individual occupancy refinement, but 'grouped' occupancy refinement: http://www.phenix-online.org/documentation/refinement.htm#anch106 HTH Peter 2008/2/6, Paul Adams :

...

Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that this is not "usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology

Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

Rife, Christopher L

5:14 p.m.

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se And, if I use "group_occupancy" plus H's, then all the residues refined to an occ of 0.99! ATOM 30 N ILE A 3 29.050 25.113 4.183 0.99 8.48 N It's fairly confusing... In the end it seemed that the best option was to not include any occupancy refinement... Chris -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Peter Zwart Sent: Wednesday, February 06, 2008 9:02 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions I guess what is needed is not individual occupancy refinement, but 'grouped' occupancy refinement: http://www.phenix-online.org/documentation/refinement.htm#anch106 HTH Peter 2008/2/6, Paul Adams :

...

Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that this is not "usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology

Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

5:25 p.m.

Hi Christopher,

...

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se

This should never happen and now I'm confused myself. To find out what is wrong, I need to reproduce it on my computer. Is it possible that you send me the data and model along with the exact command you used; I promise to keep it confidential. In this case I could have a closer look and tell exactly what's happening.

...

And, if I use "group_occupancy" plus H's, then all the residues refined to an occ of 0.99! ATOM 30 N ILE A 3 29.050 25.113 4.183 0.99 8.48 N

This is not surprising. As I wrote in my previous email in group occupancy refinement you refine one occupancy per group of atoms and this occupancy is not constrained, so it can refine to any value that the data suggests. Pavel.

Schubert, Carsten [PRDUS]

5:29 p.m.

Hi Chris, to my knowledge SHELX is the only programs available wich handles these situations correctly. Since you have very good resolution SHELX may be (arguably) the better choice at this point until Pavel is done with the implementation. Cheers, Carsten

...

-----Original Message----- From: [email protected] [mailto:[email protected]]On Behalf Of Rife, Christopher L Sent: Wednesday, February 06, 2008 12:15 PM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se

And, if I use "group_occupancy" plus H's, then all the residues refined to an occ of 0.99! ATOM 30 N ILE A 3 29.050 25.113 4.183 0.99 8.48 N

It's fairly confusing...

In the end it seemed that the best option was to not include any occupancy refinement...

Chris

-----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Peter Zwart Sent: Wednesday, February 06, 2008 9:02 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

I guess what is needed is not individual occupancy refinement, but 'grouped' occupancy refinement:

http://www.phenix-online.org/documentation/refinement.htm#anch106

HTH

Peter

...
Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that

2008/2/6, Paul Adams : this is not

...
...
"usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology

Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

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Rife, Christopher L

5:38 p.m.

Yes, we did the refinement originally in shelx, but wound up with lots of NPD atoms, which is why we're trying some other options. So far we haven't been able to adjust the parameters in shelx sufficiently to correct the problems. Regards, Chris -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Schubert, Carsten [PRDUS] Sent: Wednesday, February 06, 2008 9:30 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions Hi Chris, to my knowledge SHELX is the only programs available wich handles these situations correctly. Since you have very good resolution SHELX may be (arguably) the better choice at this point until Pavel is done with the implementation. Cheers, Carsten

...

-----Original Message----- From: [email protected] [mailto:[email protected]]On Behalf Of Rife, Christopher L Sent: Wednesday, February 06, 2008 12:15 PM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se

And, if I use "group_occupancy" plus H's, then all the residues refined to an occ of 0.99! ATOM 30 N ILE A 3 29.050 25.113 4.183 0.99 8.48 N

It's fairly confusing...

In the end it seemed that the best option was to not include any occupancy refinement...

Chris

-----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Peter Zwart Sent: Wednesday, February 06, 2008 9:02 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

I guess what is needed is not individual occupancy refinement, but 'grouped' occupancy refinement:

http://www.phenix-online.org/documentation/refinement.htm#anch106

HTH

Peter

...
Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that

2008/2/6, Paul Adams : this is not

...
...
"usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology

Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

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Pavel Afonine

5:46 p.m.

I will start implementing constrained group occupancy refinement today. It may take a few days and hopefully it will be available in one of the next versions of phenix.refine. Pavel. Rife, Christopher L wrote:

...

Yes, we did the refinement originally in shelx, but wound up with lots of NPD atoms, which is why we're trying some other options. So far we haven't been able to adjust the parameters in shelx sufficiently to correct the problems.

Regards, Chris

-----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Schubert, Carsten [PRDUS] Sent: Wednesday, February 06, 2008 9:30 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

Hi Chris,

to my knowledge SHELX is the only programs available wich handles these situations correctly. Since you have very good resolution SHELX may be (arguably) the better choice at this point until Pavel is done with the implementation.

Cheers,

Carsten

...
-----Original Message----- From: [email protected] [mailto:[email protected]]On Behalf Of Rife, Christopher L Sent: Wednesday, February 06, 2008 12:15 PM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se

And, if I use "group_occupancy" plus H's, then all the residues refined to an occ of 0.99! ATOM 30 N ILE A 3 29.050 25.113 4.183 0.99 8.48 N

It's fairly confusing...

In the end it seemed that the best option was to not include any occupancy refinement...

Chris

-----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Peter Zwart Sent: Wednesday, February 06, 2008 9:02 AM To: PHENIX user mailing list Subject: Re: [phenixbb] A few questions

I guess what is needed is not individual occupancy refinement, but 'grouped' occupancy refinement:

http://www.phenix-online.org/documentation/refinement.htm#anch106

HTH

Peter

2008/2/6, Paul Adams :

...
Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I

doesn't make

...
chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with

refmac5 and/or

...
...
...
shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that

this is not

...
...
"usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different

occupancies-

...
...
...
is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B

conf should

...
...
...
also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you

should expect

...
...
refining occupancies of atoms in alternative

conformations: the sum of

...
...
occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they

ride on bonded

...
...
atoms during coordinates refinement (riding model,

distances X-H do

...
...
not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H

atoms for this

...
...
analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology

Building 64, Room 248 Tel: 510-486-4225, Fax: 510-486-5909 http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. --

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

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Pavel Afonine

7:48 p.m.

Hi Chris, thanks for the data. I'm looking at your parameters file (test2). In the strategy keyword: strategy = *individual_sites rigid_body *individual_adp group_adp tls \ individual_occupancies *group_occupancies group_anomalous I see you are using group_occupancies which means that one occupancy per residue will be refined (unless you provide your own selections to define the groups). As I mentioned before, group_occupancies does not use any constraints and it is not aware of alternative conformations. This is why in the example below the sum of occupancies is not 1.0. I think this is not what you want to do. I will add the constrains to group_occupancies option so you get equal occupancies within each conformer and the sum of occupancies computed for all conformers is 1.0. Currently, if you choose individual_occupancies then you will get the constraints, that is the sum of occupancies for "523 SE AMSE A 32" and "530 SE BMSE A 32" will be one, but the occupancies for atoms within each conformer will be different, which is, as Paul correctly pointed out, not 100% right. I will keep you posted about the progress. Pavel.

...

In that case, then the occ of alt conf's tends to refine to greater than 1: ATOM 523 SE AMSE A 32 23.654 11.792 6.216 0.56 14.04 Se ATOM 530 SE BMSE A 32 23.570 12.284 6.796 0.46 13.91 Se

David Garboczi

7 Feb 7 Feb

8:07 p.m.

New subject: picking rfree

I am working with a dataset that I believe to be P21 even though the beta angle is 89.9. I picked the rfree set with phenix and expected it to pick the set in the orthorhombic set, because use_lattice_symmetry= True is the default. But the log file did not reflect that phenix recognized the almost orthorhombic symmetry. I'd like to know if it did pick the rfree set in the higher symmetry or not. thanks, Dave -- David N. Garboczi, PhD Phone: 301-496-4773 Investigator, Structural Biology Section (SBS) Laboratory of Immunogenetics (LIG) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH) Twinbrook 2/Room 110 12441 Parklawn Drive Rockville, Maryland 20852-1742 Fax: 301-402-0284 Email: [email protected] The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. The National Institute of Allergy and Infectious Diseases (NIAID) shall not accept liability for any statement made that are the sender's own and not expressly made on behalf of the NIAID by one of its representatives.

Mischa Machius

8:11 p.m.

New subject: picking rfree

We just had a monoclinic case with a beta angle of 90.02! That's just to show that such things do indeed occur. Best - MM On Feb 7, 2008, at 2:07 PM, David Garboczi wrote:

...

I am working with a dataset that I believe to be P21 even though the beta angle is 89.9. I picked the rfree set with phenix and expected it to pick the set in the orthorhombic set, because use_lattice_symmetry= True is the default.

But the log file did not reflect that phenix recognized the almost orthorhombic symmetry. I'd like to know if it did pick the rfree set in the higher symmetry or not.

thanks,

Dave

-- David N. Garboczi, PhD Phone: 301-496-4773 Investigator, Structural Biology Section (SBS) Laboratory of Immunogenetics (LIG) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH) Twinbrook 2/Room 110 12441 Parklawn Drive Rockville, Maryland 20852-1742 Fax: 301-402-0284 Email: [email protected]

The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. The National Institute of Allergy and Infectious Diseases (NIAID) shall not accept liability for any statement made that are the sender's own and not expressly made on behalf of the NIAID by one of its representatives. _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

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

Ralf W. Grosse-Kunstleve

8:24 p.m.

New subject: picking rfree

Hi Dave,

...

I am working with a dataset that I believe to be P21 even though the beta angle is 89.9. I picked the rfree set with phenix and expected it to pick the set in the orthorhombic set, because use_lattice_symmetry= True is the default.

But the log file did not reflect that phenix recognized the almost orthorhombic symmetry.

Yes, that's (unfortunately) true. The whole calculation is done in a black box which doesn't report back the lattice symmetry it used internally.

...

I'd like to know if it did pick the rfree set in the higher symmetry or not.

An indirect way to find out is to force the higher symmetry. phenix.refine will merge the R-free set and complain if symmetry-equivalent flags are incompatible. To do this: phenix.refine your.mtz your.pdb --space-group=p222 --unit-cell="21.937 4.866 23.476 90 90 90" refinement.input.symmetry_safety_check=warning It will be best to use the correct a,b,c unit cell parameters instead of the dummy values. If something is wrong you will see a message like this: Checking symmetry-equivalent R-free flags for consistency: Sorry: Incompatible symmetry-equivalent R-free flags: incompatible flags for hkl = (1, 0, 5) If it runs beyond the point of analyzing the input "X-ray data" the flags are what you are hoping for. Ralf

David Garboczi

9:02 p.m.

New subject: picking rfree

...

Hi Dave,

...
I am working with a dataset that I believe to be P21 even though the beta angle is 89.9. I picked the rfree set with phenix and expected it to pick the set in the orthorhombic set, because use_lattice_symmetry= True is the default.

But the log file did not reflect that phenix recognized the almost orthorhombic symmetry.

Yes, that's (unfortunately) true. The whole calculation is done in a black box which doesn't report back the lattice symmetry it used internally.

...
I'd like to know if it did pick the rfree set in the higher symmetry or not.

An indirect way to find out is to force the higher symmetry. phenix.refine will merge the R-free set and complain if symmetry-equivalent flags are incompatible.

To do this:

phenix.refine your.mtz your.pdb --space-group=p222 --unit-cell="21.937 4.866 23.476 90 90 90" refinement.input.symmetry_safety_check=warning

It will be best to use the correct a,b,c unit cell parameters instead of the dummy values.

If something is wrong you will see a message like this:

Checking symmetry-equivalent R-free flags for consistency: Sorry: Incompatible symmetry-equivalent R-free flags: incompatible flags for hkl = (1, 0, 5)

If it runs beyond the point of analyzing the input "X-ray data" the flags are what you are hoping for.

Ralf _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

Ralf, That worked. I forced the p222 and got this line in the file. Checking symmetry-equivalent R-free flags for consistency: OK thanks, Dave -- David N. Garboczi, PhD Phone: 301-496-4773 Investigator, Structural Biology Section (SBS) Laboratory of Immunogenetics (LIG) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH) Twinbrook 2/Room 110 12441 Parklawn Drive Rockville, Maryland 20852-1742 Fax: 301-402-0284 Email: [email protected] The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. The National Institute of Allergy and Infectious Diseases (NIAID) shall not accept liability for any statement made that are the sender's own and not expressly made on behalf of the NIAID by one of its representatives.

Pavel Afonine

6 Feb 6 Feb

5:16 p.m.

Hi Paul, currently phenix.refine can refine occupancies as: individual, constrained individual and group. - In individual occupancy refinement occupancy of each atom is refined individually. - In constrained individual occupancy refinement occupancies of each atom are refined "individually" but it is made sure that the sum of them is one (for each atom, not group of atoms). - In group occupancy refinement you can refine one occupancy per selected group of atoms, without any constrains. What you mention is "constrained group occupancy refinement" where you refine one "individual" occupancy per group of atoms making sure that the sum of them is one. Unfortunately this is not implemented. This is on the top of my to-do list, so hopefully we will have it soon. Pavel. Paul Adams wrote:

...

Hi Pavel,

the occupancy should be the same for all of the atoms in an alternate conformation (grouped occupancy refinement). I doesn't make chemical sense for bonded atoms to have different occupancies.

Cheers, Paul

On Feb 6, 2008, at 8:56 AM, Pavel Afonine wrote:

...
Hi Christopher,

thanks for your questions!

...
We are working on a refinement at 1.14A which was suffering from lots of NPD atoms when refined anisotropically with refmac5 and/or shelx.

The implementation of anisotropic B-factors refinement in phenix.refine should never lead to non-positive definite ADP matrices or any "instability" in refinement.

...
1. In our model we have a few solvent ligands, and these show up in the log file as: Unusual residues: {'EDO': 11, ' CL': 1, 'SO4': 1} Classifications: {'undetermined': 13, 'water': 437} We've used a cif file for EDO with H's when needed, but still see this message. Is this normal? What is unusual?

Yes, it is normal. In this context "unusual" means that this is not "usual amino acid, water, dna/rna fragment".

...
2. When choosing "indivdual_occupancies" for the refinement, all the atoms within an alt-conf are refined to different occupancies- is this expected? It seems that all atoms in the A conf should have the same value, and the occ of all atoms in the B conf should also be the same. Instead, we see this: ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ATOM 233 CB AGLU A 28 24.851 7.269 3.672 0.80 9.27 C ATOM 234 CG AGLU A 28 24.978 6.767 2.259 0.66 12.91 C ATOM 235 CD AGLU A 28 23.662 6.717 1.554 1.00 16.51 C ATOM 236 OE1AGLU A 28 22.874 7.667 1.757 0.71 18.78 O ATOM 237 OE2AGLU A 28 23.429 5.733 0.803 0.60 16.77 O ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C ATOM 241 CB BGLU A 28 24.794 7.230 3.713 0.20 6.48 C ATOM 242 CG BGLU A 28 24.877 6.766 2.280 0.34 6.55 C ATOM 243 CD BGLU A 28 23.536 6.702 1.598 0.00 7.53 C ATOM 244 OE1BGLU A 28 23.497 7.014 0.381 0.29 7.85 O ATOM 245 OE2BGLU A 28 22.543 6.341 2.280 0.40 8.40 O

This is correct behavior and this is exactly what you should expect refining occupancies of atoms in alternative conformations: the sum of occupancies of conformation A and B must be one. So, in your example above:

ATOM 232 CA AGLU A 28 26.163 7.163 4.422 0.77 6.73 C ... ATOM 240 CA BGLU A 28 26.140 7.156 4.424 0.23 6.33 C

the total occupancy is: 0.77+0.23=1 Same for other atoms.

...
3. Adding hydrogens during anisotropic refinement results in the Parvati server giving "Illegal Biso" errors for many of the hydrogens.

The H atoms at normal resolutions (not subatomic resolution) are treated as a special case. For example (default behavior), they ride on bonded atoms during coordinates refinement (riding model, distances X-H do not change), we refine one occupancy and isotropic B-factor per all H atoms in your molecule, etc. I don't know what exactly "Illegal Biso" means in Parvati server, but most likely you want to exclude H atoms for this analysis.

Just a suggestion: at resolution 1.4A you can try to change the default behavior for H refinement to this:

hydrogens { refine_adp = *one_b_per_residue one_b_per_molecule individual refine_occupancies = *one_q_per_residue one_q_per_molecule individual }

Please let us know if you have any questions or problems! Pavel.

_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

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David Garboczi
Mischa Machius
Paul Adams
Pavel Afonine
Pavel Afonine
Peter Zwart
Ralf W. Grosse-Kunstleve
Rife, Christopher L
Schubert, Carsten [PRDUS]

A few questions

Rife, Christopher L

Pavel Afonine

Paul Adams

Peter Zwart

Rife, Christopher L

Pavel Afonine

Schubert, Carsten [PRDUS]

Rife, Christopher L

Pavel Afonine

Pavel Afonine

David Garboczi

Mischa Machius

Ralf W. Grosse-Kunstleve

David Garboczi

Pavel Afonine

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