Hi Xavier, I remember we had a similar case in January and had a long conversation about it between Nigel, Oleg and me. The solution at that time was to update Phenix library with that non-standard amino-acid-like entity so that Phenix produces mmcif file that is legible by PDB. In your case it does seem you have "SER plus something" as the modified residue. I think the easiest way for you to proceed is to follow Nigel's suggestion. Pavel On 4/20/22 14:10, Nigel Moriarty wrote:

Xavier

I'm sure others can solve this problem but it adds to my point that if you have a covalently bound ligand to an amino acid that does not change the main chain, it is generally "better" to maintain the, in this case SER, and generate the ligand and links to the side chain.

Just one of a number of reasons.

Cheers

Nigel

--- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709     Email : [email protected] Fax   : 510-486-5909      Web  : CCI.LBL.gov http://CCI.LBL.gov ORCID : orcid.org/0000-0001-8857-9464 https://orcid.org/0000-0001-8857-9464

On Wed, Apr 20, 2022 at 2:02 PM Xavier Brazzolotto wrote:

Dear Phenix users,

I don’t know if my problem is related to Phenix but for information I’m running Phenix 1.20.1-4487 under MacOS 12.3.1.

I’ve finalized a structure where a ligand covalently modified the protein.

I’ve generated the modified residue (named SLG for serine modified by ligand). For this I’ve generated the molecules in SMILES and used eLBOW to generate the restraints. Then I’ve modified the cif file defining the molecule as a L-peptide and replacing the atom names of the Serine part (CA, CB, OG, C, O, N, and OXT) In coot (from CCP4 : 0.9.6 EL), I’ve used the modified cif file and it allowed merging of the modified residue into the polypeptide chain as expected and further refinements went without any issue in Phenix (providing the modified cif file of course). Everything seems well interpreted. So far so good.

However, now I would like to validate the structure and both Phenix validation tool and the PDB web server do not accept the final cif file.

Checking this file I’ve noticed that the protein seems split into 3 pieces (chain A, first residue up to the one before the modified residue; chain B the modified residue by itself described as HETATM and chain C the rest of the polypeptide up to the C-ter). The PDB file presents only one chain A for the whole protein with the modified residue...

I don’t know if this is an issue with Phenix generating this final cif file in this specific case or if I need to modify this final file by hand ?

Any help is welcome. Thanks

Xavier

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Xavier Paul's point is very valid because the "Prepare for Deposition" step is what generates the sequence (which is the crucial point here) for deposition. However, because you have "created" a new amino acid, there will still be issues as highlighted by Pavel. It is a corner case. One small addition point is that SLG is already taken in the PDB Ligand list. There are tools in Phenix to find an used code. Can you re-engineer it with SER+ligand? This will solve the problem using the current Phenix version. I can help with the details if needed. Cheers Nigel --- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : [email protected] Fax : 510-486-5909 Web : CCI.LBL.gov ORCID : orcid.org/0000-0001-8857-9464 On Wed, Apr 20, 2022 at 5:02 PM Paul Adams wrote:

Please also remember that you need to run “Prepare model for PDB deposition” (in the GUI under "PDB Deposition") on the mmCIF file you get from phenix.refine. This provides important information that is required for the deposition at the PDB.

On Apr 20, 2022, at 1:58 PM, Xavier Brazzolotto wrote:

Dear Phenix users,

I don’t know if my problem is related to Phenix but for information I’m running Phenix 1.20.1-4487 under MacOS 12.3.1.

I’ve finalized a structure where a ligand covalently modified the protein.

I’ve generated the modified residue (named SLG for serine modified by ligand). For this I’ve generated the molecules in SMILES and used eLBOW to generate the restraints. Then I’ve modified the cif file defining the molecule as a L-peptide and replacing the atom names of the Serine part (CA, CB, OG, C, O, N, and OXT) In coot (from CCP4 : 0.9.6 EL), I’ve used the modified cif file and it allowed merging of the modified residue into the polypeptide chain as expected and further refinements went without any issue in Phenix (providing the modified cif file of course). Everything seems well interpreted. So far so good.

However, now I would like to validate the structure and both Phenix validation tool and the PDB web server do not accept the final cif file.

Checking this file I’ve noticed that the protein seems split into 3 pieces (chain A, first residue up to the one before the modified residue; chain B the modified residue by itself described as HETATM and chain C the rest of the polypeptide up to the C-ter). The PDB file presents only one chain A for the whole protein with the modified residue...

I don’t know if this is an issue with Phenix generating this final cif file in this specific case or if I need to modify this final file by hand ?

Any help is welcome. Thanks

Xavier

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

-- Paul Adams (he/him/his) Associate Laboratory Director for Biosciences, LBL ( https://biosciences.lbl.gov/leadership/) Principal Investigator, Computational Crystallography Initiative, LBL ( https://cci.lbl.gov) Vice President for Technology, the Joint BioEnergy Institute ( http://www.jbei.org) Principal Investigator, ALS-ENABLE, Advanced Light Source ( https://als-enable.lbl.gov) Division Deputy for Biosciences, Advanced Light Source ( https://als.lbl.gov) Laboratory Research Manager, ENIGMA Science Focus Area ( http://enigma.lbl.gov) Adjunct Professor, Department of Bioengineering, UC Berkeley ( http://bioeng.berkeley.edu) Member of the Graduate Group in Comparative Biochemistry, UC Berkeley ( http://compbiochem.berkeley.edu)

Building 33, Room 250 Building 978, Room 4126 Building 977, Room 268 Tel: 1-510-486-4225 http://cci.lbl.gov/paul ORCID: 0000-0001-9333-8219

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 33R0345 Berkeley, CA 94720, USA.

Executive Assistant: Michael Espinosa [ [email protected] ] [ 1-510-333-6788 ] Phenix Consortium: Ashley Dawn [ [email protected] ][ 1-510-486-5455 ]

--

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I’ve re-processed the structure separating the SER residue from the ligand part. Now I have independent ligand. In the « Custom Geometry Restraints » I’ve defined the bond between OG and the carbon atom of the ligand and I’ve defined the angles (I’ve used the values from the previously determined eLBOW run off the SER-bound ligand complex), saved the restraints and launched the refinement. At a first look it was processed correctly and the final cif file has now the whole protein in Chain A. I’ve used prepare PDB deposition using the FASTA sequence of the protein (wonder if I have to provide the ligand CIF file and add more options) and ran phenix.get_pdb_validation : the report looks ok for protein and some other basic ligands (sugars, buffer, glycerol, etc...) but the ligand of interest was not processed (EDS FAILED...). In the PDB file, all these extra ligands are also in Chain A, with water in chain B. If I try the validation through the website (PDBe@EBI) with both cif files from the Refine or the Prepare_PDB_Deposition process, both seem to crash the server as it takes forever without Finalizing... I wonder if I am missing something… Maybe declaration of removal of atoms : HG bound to OG in SER or/and removal of one H from the carbon of the ligand involved in the bond ? Xavier

Le 21 avr. 2022 à 08:06, Xavier Brazzolotto a écrit :

Thank you for your feedback.

@Paul, I’ve run the « Prepare model for deposition » with the option modified residue (SLG). Not sure it will change if I change the name as it is already the PDB database, but I will give it another try.

I think that I will have to describe only the ligand and add some parameters restricting distance and angles between the OG of SER and the ligand, I think this is right way. @ Nigel, is that what you mean with « details » ? If you have any other « tips/tricks » they are welcome.

Best Xavier

...

Le 21 avr. 2022 à 02:47, Nigel Moriarty mailto:[email protected]> a écrit :

Xavier

Paul's point is very valid because the "Prepare for Deposition" step is what generates the sequence (which is the crucial point here) for deposition. However, because you have "created" a new amino acid, there will still be issues as highlighted by Pavel. It is a corner case.

One small addition point is that SLG is already taken in the PDB Ligand list. There are tools in Phenix to find an used code.

Can you re-engineer it with SER+ligand? This will solve the problem using the current Phenix version. I can help with the details if needed.

Cheers

Nigel

--- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : [email protected] mailto:[email protected] Fax : 510-486-5909 Web : CCI.LBL.gov http://cci.lbl.gov/ ORCID : orcid.org/0000-0001-8857-9464 https://orcid.org/0000-0001-8857-9464

On Wed, Apr 20, 2022 at 5:02 PM Paul Adams mailto:[email protected]> wrote:

Please also remember that you need to run “Prepare model for PDB deposition” (in the GUI under "PDB Deposition") on the mmCIF file you get from phenix.refine. This provides important information that is required for the deposition at the PDB.

...

On Apr 20, 2022, at 1:58 PM, Xavier Brazzolotto mailto:[email protected]> wrote:

Dear Phenix users,

I don’t know if my problem is related to Phenix but for information I’m running Phenix 1.20.1-4487 under MacOS 12.3.1.

I’ve finalized a structure where a ligand covalently modified the protein.

I’ve generated the modified residue (named SLG for serine modified by ligand). For this I’ve generated the molecules in SMILES and used eLBOW to generate the restraints. Then I’ve modified the cif file defining the molecule as a L-peptide and replacing the atom names of the Serine part (CA, CB, OG, C, O, N, and OXT) In coot (from CCP4 : 0.9.6 EL), I’ve used the modified cif file and it allowed merging of the modified residue into the polypeptide chain as expected and further refinements went without any issue in Phenix (providing the modified cif file of course). Everything seems well interpreted. So far so good.

However, now I would like to validate the structure and both Phenix validation tool and the PDB web server do not accept the final cif file.

Checking this file I’ve noticed that the protein seems split into 3 pieces (chain A, first residue up to the one before the modified residue; chain B the modified residue by itself described as HETATM and chain C the rest of the polypeptide up to the C-ter). The PDB file presents only one chain A for the whole protein with the modified residue...

I don’t know if this is an issue with Phenix generating this final cif file in this specific case or if I need to modify this final file by hand ?

Any help is welcome. Thanks

Xavier

_______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected] mailto:[email protected] -- Paul Adams (he/him/his) Associate Laboratory Director for Biosciences, LBL (https://biosciences.lbl.gov/leadership/ https://biosciences.lbl.gov/leadership/) Principal Investigator, Computational Crystallography Initiative, LBL (https://cci.lbl.gov https://cci.lbl.gov/) Vice President for Technology, the Joint BioEnergy Institute (http://www.jbei.org http://www.jbei.org/) Principal Investigator, ALS-ENABLE, Advanced Light Source (https://als-enable.lbl.gov https://als-enable.lbl.gov/) Division Deputy for Biosciences, Advanced Light Source (https://als.lbl.gov https://als.lbl.gov/) Laboratory Research Manager, ENIGMA Science Focus Area (http://enigma.lbl.gov http://enigma.lbl.gov/) Adjunct Professor, Department of Bioengineering, UC Berkeley (http://bioeng.berkeley.edu http://bioeng.berkeley.edu/) Member of the Graduate Group in Comparative Biochemistry, UC Berkeley (http://compbiochem.berkeley.edu http://compbiochem.berkeley.edu/)

Building 33, Room 250 Building 978, Room 4126 Building 977, Room 268 Tel: 1-510-486-4225 http://cci.lbl.gov/paul http://cci.lbl.gov/paul ORCID: 0000-0001-9333-8219

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 33R0345 Berkeley, CA 94720, USA.

Executive Assistant: Michael Espinosa [ [email protected] mailto:[email protected] ] [ 1-510-333-6788 ] Phenix Consortium: Ashley Dawn [ [email protected] mailto:[email protected] ][ 1-510-486-5455 ]

--

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I recall now how I used to add the glycans before the Carbohydrate Module in Coot. I have to make the cif file with the putative O atom of SER OG and remove it before making the bond. I will try that, it will certainly correct my geometry issue but I am still not sure for the final file and validation. Fingers crossed...

Le 21 avr. 2022 à 17:09, Xavier Brazzolotto a écrit :

After some careful inspection. The geometry on the C atom of the ligand is weird, I don’t get something close to tetrahedral (or similar). Probably some angles are missing or I did something wrong with the ligand cif file. Not fixed yet

...

Le 21 avr. 2022 à 13:39, Xavier Brazzolotto mailto:[email protected]> a écrit :

I’ve re-processed the structure separating the SER residue from the ligand part. Now I have independent ligand. In the « Custom Geometry Restraints » I’ve defined the bond between OG and the carbon atom of the ligand and I’ve defined the angles (I’ve used the values from the previously determined eLBOW run off the SER-bound ligand complex), saved the restraints and launched the refinement. At a first look it was processed correctly and the final cif file has now the whole protein in Chain A.

I’ve used prepare PDB deposition using the FASTA sequence of the protein (wonder if I have to provide the ligand CIF file and add more options) and ran phenix.get_pdb_validation : the report looks ok for protein and some other basic ligands (sugars, buffer, glycerol, etc...) but the ligand of interest was not processed (EDS FAILED...). In the PDB file, all these extra ligands are also in Chain A, with water in chain B.

If I try the validation through the website (PDBe@EBI) with both cif files from the Refine or the Prepare_PDB_Deposition process, both seem to crash the server as it takes forever without Finalizing...

I wonder if I am missing something… Maybe declaration of removal of atoms : HG bound to OG in SER or/and removal of one H from the carbon of the ligand involved in the bond ?

Xavier

...

Le 21 avr. 2022 à 08:06, Xavier Brazzolotto mailto:[email protected]> a écrit :

Thank you for your feedback.

@Paul, I’ve run the « Prepare model for deposition » with the option modified residue (SLG). Not sure it will change if I change the name as it is already the PDB database, but I will give it another try.

I think that I will have to describe only the ligand and add some parameters restricting distance and angles between the OG of SER and the ligand, I think this is right way. @ Nigel, is that what you mean with « details » ? If you have any other « tips/tricks » they are welcome.

Best Xavier

...

Le 21 avr. 2022 à 02:47, Nigel Moriarty mailto:[email protected]> a écrit :

Xavier

Paul's point is very valid because the "Prepare for Deposition" step is what generates the sequence (which is the crucial point here) for deposition. However, because you have "created" a new amino acid, there will still be issues as highlighted by Pavel. It is a corner case.

One small addition point is that SLG is already taken in the PDB Ligand list. There are tools in Phenix to find an used code.

Can you re-engineer it with SER+ligand? This will solve the problem using the current Phenix version. I can help with the details if needed.

Cheers

Nigel

--- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : [email protected] mailto:[email protected] Fax : 510-486-5909 Web : CCI.LBL.gov http://cci.lbl.gov/ ORCID : orcid.org/0000-0001-8857-9464 https://orcid.org/0000-0001-8857-9464

On Wed, Apr 20, 2022 at 5:02 PM Paul Adams mailto:[email protected]> wrote:

Please also remember that you need to run “Prepare model for PDB deposition” (in the GUI under "PDB Deposition") on the mmCIF file you get from phenix.refine. This provides important information that is required for the deposition at the PDB.

...

On Apr 20, 2022, at 1:58 PM, Xavier Brazzolotto mailto:[email protected]> wrote:

Dear Phenix users,

I don’t know if my problem is related to Phenix but for information I’m running Phenix 1.20.1-4487 under MacOS 12.3.1.

I’ve finalized a structure where a ligand covalently modified the protein.

I’ve generated the modified residue (named SLG for serine modified by ligand). For this I’ve generated the molecules in SMILES and used eLBOW to generate the restraints. Then I’ve modified the cif file defining the molecule as a L-peptide and replacing the atom names of the Serine part (CA, CB, OG, C, O, N, and OXT) In coot (from CCP4 : 0.9.6 EL), I’ve used the modified cif file and it allowed merging of the modified residue into the polypeptide chain as expected and further refinements went without any issue in Phenix (providing the modified cif file of course). Everything seems well interpreted. So far so good.

However, now I would like to validate the structure and both Phenix validation tool and the PDB web server do not accept the final cif file.

Checking this file I’ve noticed that the protein seems split into 3 pieces (chain A, first residue up to the one before the modified residue; chain B the modified residue by itself described as HETATM and chain C the rest of the polypeptide up to the C-ter). The PDB file presents only one chain A for the whole protein with the modified residue...

I don’t know if this is an issue with Phenix generating this final cif file in this specific case or if I need to modify this final file by hand ?

Any help is welcome. Thanks

Xavier

_______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected] mailto:[email protected] -- Paul Adams (he/him/his) Associate Laboratory Director for Biosciences, LBL (https://biosciences.lbl.gov/leadership/ https://biosciences.lbl.gov/leadership/) Principal Investigator, Computational Crystallography Initiative, LBL (https://cci.lbl.gov https://cci.lbl.gov/) Vice President for Technology, the Joint BioEnergy Institute (http://www.jbei.org http://www.jbei.org/) Principal Investigator, ALS-ENABLE, Advanced Light Source (https://als-enable.lbl.gov https://als-enable.lbl.gov/) Division Deputy for Biosciences, Advanced Light Source (https://als.lbl.gov https://als.lbl.gov/) Laboratory Research Manager, ENIGMA Science Focus Area (http://enigma.lbl.gov http://enigma.lbl.gov/) Adjunct Professor, Department of Bioengineering, UC Berkeley (http://bioeng.berkeley.edu http://bioeng.berkeley.edu/) Member of the Graduate Group in Comparative Biochemistry, UC Berkeley (http://compbiochem.berkeley.edu http://compbiochem.berkeley.edu/)

Building 33, Room 250 Building 978, Room 4126 Building 977, Room 268 Tel: 1-510-486-4225 http://cci.lbl.gov/paul http://cci.lbl.gov/paul ORCID: 0000-0001-9333-8219

Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 33R0345 Berkeley, CA 94720, USA.

Executive Assistant: Michael Espinosa [ [email protected] mailto:[email protected] ] [ 1-510-333-6788 ] Phenix Consortium: Ashley Dawn [ [email protected] mailto:[email protected] ][ 1-510-486-5455 ]

--

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