Dear Maike, As you might we have been developing  macromolecular  refinements methods based on linear scaling QM approaches (Borbulevych et al. Acta Cryst., 2014, D70, 1233-1247; Acta Cryst., 2016, D72, 586-598). One benefit of the QM refinement in PHENIX is that it objectively reveal the ligand geometry and does not rely on provided CIF. You can contact [email protected] to obtain download instructions of our package. Alternatively, you can also provide your input files  so we would try out your structure here. Oleg Oleg Borbulevych, Ph.D. Staff Scientist QuantumBio, Inc. 2790 W. College Ave. State College, PA 16801 E-mail: [email protected], [email protected] Linkedin Professional Profile: http://www.linkedin.com/pub/oleg-borbulevych/22/454/b80 -----Original Message----- From: "Maike Bublitz" Sent: Monday, February 27, 2017 12:45pm To: "Tanner, John J." Cc: "[email protected]" Subject: Re: [phenixbb] Phenix.refine ignores ligand planarity restraint _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected] John, thanks for the suggestion - I’ve tried both 0.001 and zero, as yet without success. /Maike On 27.Feb 2017, at 17:12 , Tanner, John J. mailto:[email protected]> wrote: You could try setting the deviation from planarity parameter to a very small value (maybe zero?).  It is the number after the atom name: NAP plan-1    C6A 0.020 On Feb 27, 2017, at 11:05 AM, Maike Bublitz mailto:[email protected]> wrote: Dear all, this has probably been discussed many times before, but I just can’t seem to find a solution for my current problem with phenix.refine: I am trying to refine a protein structure with a rather complex ligand. I’ve generated a .cif file for the ligand with eLBOW, including planarity restraints for its aromatic ring systems. However, phenix.refine keeps on distorting the 6-ring in my indole group, although all respective atoms are included in the plane definition. At a resolution of 3.0 A, I’d very much prefer to impose an ideal planar structure on this ligand. How can I convince Phenix to not distort my ligand's aromatic rings? Many thanks for your kind suggestions. Best regards, Maike ************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk/ _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]mailto:[email protected] _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]

Dear all, thank you again for all your suggestions. Here’s what I’ve tried, without success: 1) reduce deviation from planarity restraints to very small value or zero -> ligand still gets distorted in refinement 2) Set all bond angle restraints to ideal values and checked sums of angles in planar rings -> ligand still gets distorted in refinement 3) Used phenix.elbow with - -final-geometry on a pdb of a planar ligand —> ligand still gets distorted in refinement 4) Used the grade server (grade.globalphasing.orghttp://grade.globalphasing.org) to generate cif file from SMILES string—> phenix.refine stops with message “Unknown file format: grade-ligand.cif”. Does anyone have another suggestion, except for installing an extra QM package? Best, Maike On 27.Feb 2017, at 17:24 , Dale Tronrud mailto:[email protected]> wrote: You could also check that the other restraints in your cif are compatible with planarity. If the sum of the three bond angles for a planar atom is less than 360 deg your angle restraints will be fighting with your planarity. You also have to check the sum of the internal angles of your planar rings. If I recall correctly the sum of the internal angles must equal n*180 - 360 for the ring to be flat. Dale Tronrud On 2/27/2017 9:05 AM, Maike Bublitz wrote: Dear all, this has probably been discussed many times before, but I just can’t seem to find a solution for my current problem with phenix.refine: I am trying to refine a protein structure with a rather complex ligand. I’ve generated a .cif file for the ligand with eLBOW, including planarity restraints for its aromatic ring systems. However, phenix.refine keeps on distorting the 6-ring in my indole group, although all respective atoms are included in the plane definition. At a resolution of 3.0 A, I’d very much prefer to impose an ideal planar structure on this ligand. How can I convince Phenix to not distort my ligand's aromatic rings? Many thanks for your kind suggestions. Best regards, Maike ************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected] _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected] ************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk

Excellent point! Have a look at *.geo file that lists absolutely all geometry restraints that are used in refinement (bonds, angles, torsions, planes, chirals, non-bonded, ncs, etc..). If the plane in question is not listed there then we will need to investigate why is that. Pavel On 2/28/17 09:01, Tim Gruene wrote:

Dear Maike,

did you read the log file to see whether any of your restraints get acknowledged by phenix.refine? There can be many reasons why they are not, e.g. mismatching atom names, mismatching residue abbreviation, ...

Regards, Tim

On Tuesday 28 February 2017 04:44:43 PM Maike Bublitz wrote:

...

Dear all,

thank you again for all your suggestions. Here’s what I’ve tried, without success:

1) reduce deviation from planarity restraints to very small value or zero -> ligand still gets distorted in refinement

2) Set all bond angle restraints to ideal values and checked sums of angles in planar rings -> ligand still gets distorted in refinement

3) Used phenix.elbow with - -final-geometry on a pdb of a planar ligand —> ligand still gets distorted in refinement

4) Used the grade server (grade.globalphasing.orghttp://grade.globalphasing.org) to generate cif file from SMILES string—> phenix.refine stops with message “Unknown file format: grade-ligand.cif”.

Does anyone have another suggestion, except for installing an extra QM package?

Best, Maike

On 27.Feb 2017, at 17:24 , Dale Tronrud mailto:[email protected]> wrote:

You could also check that the other restraints in your cif are compatible with planarity. If the sum of the three bond angles for a planar atom is less than 360 deg your angle restraints will be fighting with your planarity. You also have to check the sum of the internal angles of your planar rings. If I recall correctly the sum of the internal angles must equal n*180 - 360 for the ring to be flat.

Dale Tronrud

On 2/27/2017 9:05 AM, Maike Bublitz wrote: Dear all,

this has probably been discussed many times before, but I just can’t seem to find a solution for my current problem with phenix.refine:

I am trying to refine a protein structure with a rather complex ligand. I’ve generated a .cif file for the ligand with eLBOW, including planarity restraints for its aromatic ring systems. However, phenix.refine keeps on distorting the 6-ring in my indole group, although all respective atoms are included in the plane definition. At a resolution of 3.0 A, I’d very much prefer to impose an ideal planar structure on this ligand.

How can I convince Phenix to not distort my ligand's aromatic rings?

Many thanks for your kind suggestions.

Best regards, Maike

************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk

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Maike One more word of advice. Try to provide eLBOW with as much information as possible. Your restraints file showed that you had used a PDB file input without, I assume, hydrogens. A SMILES string is a better choice as it has a higher chemical information content. BTW, Phil Jeffrey is correct that zero esd turns off the restraint. Also, making the esd too small is also not advisable. For example, a plane esd of 0.02 is fine, but 0.002 may be too small. As a rule of thumb, don't make the esd more than 10x smaller than the default. 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 On Tue, Feb 28, 2017 at 11:30 AM, Maike Bublitz < [email protected]> wrote:

Dear all,

as it turns out, the cif file was just fine after I had defined the planes properly. I was just accidentally using a script in which I had switched off coordinate refinement. D’Oh. My desk has a bite mark now.

Thanks, Pavel, for spotting this, and thanks to all for your suggestions.

Cheers, Maike

On 28.Feb 2017, at 17:13 , Pavel Afonine wrote:

Excellent point! Have a look at *.geo file that lists absolutely all geometry restraints that are used in refinement (bonds, angles, torsions, planes, chirals, non-bonded, ncs, etc..). If the plane in question is not listed there then we will need to investigate why is that.

Pavel

On 2/28/17 09:01, Tim Gruene wrote:

Dear Maike,

did you read the log file to see whether any of your restraints get acknowledged by phenix.refine? There can be many reasons why they are not, e.g. mismatching atom names, mismatching residue abbreviation, ...

Regards, Tim

On Tuesday 28 February 2017 04:44:43 PM Maike Bublitz wrote:

Dear all,

thank you again for all your suggestions. Here’s what I’ve tried, without success:

1) reduce deviation from planarity restraints to very small value or zero -> ligand still gets distorted in refinement

2) Set all bond angle restraints to ideal values and checked sums of angles in planar rings -> ligand still gets distorted in refinement

3) Used phenix.elbow with - -final-geometry on a pdb of a planar ligand —> ligand still gets distorted in refinement

4) Used the grade server (grade.globalphasing.orghttp://grade.globalphasing.org http://grade.globalphasing.org/) to generate cif file from SMILES string—> phenix.refine stops with message “Unknown file format: grade-ligand.cif”.

Does anyone have another suggestion, except for installing an extra QM package?

Best, Maike

On 27.Feb 2017, at 17:24 , Dale Tronrud mailto:[email protected] > wrote:

You could also check that the other restraints in your cif are compatible with planarity. If the sum of the three bond angles for a planar atom is less than 360 deg your angle restraints will be fighting with your planarity. You also have to check the sum of the internal angles of your planar rings. If I recall correctly the sum of the internal angles must equal n*180 - 360 for the ring to be flat.

Dale Tronrud

On 2/27/2017 9:05 AM, Maike Bublitz wrote: Dear all,

this has probably been discussed many times before, but I just can’t seem to find a solution for my current problem with phenix.refine:

I am trying to refine a protein structure with a rather complex ligand. I’ve generated a .cif file for the ligand with eLBOW, including planarity restraints for its aromatic ring systems. However, phenix.refine keeps on distorting the 6-ring in my indole group, although all respective atoms are included in the plane definition. At a resolution of 3.0 A, I’d very much prefer to impose an ideal planar structure on this ligand.

How can I convince Phenix to not distort my ligand's aromatic rings?

Many thanks for your kind suggestions.

Best regards, Maike

************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 lab: +44 (0)1865 613318 maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk/http://bioch.ox.ac.uk http://bioch.ox.ac.uk/

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk/

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.uk

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Maike That is a reasonable approach. Having a well minimised geometry with hydrogens has all the right ingredients. 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 On Tue, Feb 28, 2017 at 11:49 AM, Maike Bublitz < [email protected]> wrote:

Dear Nigel,

thanks for pointing this out. I had actually given eLBOW a pdb model of the ligand with hydrogens. This model came out of maestro, where I had built and energy-minimized it. In that cif file you saw, I had deleted all the hydrogens later on, because the ligand in my refinement complex didn’t have any, and in my desperation I thought that might be a problem.

Best, Maike

On 28.Feb 2017, at 19:41 , Nigel Moriarty wrote:

Maike

One more word of advice. Try to provide eLBOW with as much information as possible. Your restraints file showed that you had used a PDB file input without, I assume, hydrogens. A SMILES string is a better choice as it has a higher chemical information content.

BTW, Phil Jeffrey is correct that zero esd turns off the restraint. Also, making the esd too small is also not advisable. For example, a plane esd of 0.02 is fine, but 0.002 may be too small. As a rule of thumb, don't make the esd more than 10x smaller than the default.

Cheers

Nigel

--- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 <(510)%20486-5709> Email : [email protected] Fax : 510-486-5909 <(510)%20486-5909> Web : CCI.LBL.gov http://cci.lbl.gov/

On Tue, Feb 28, 2017 at 11:30 AM, Maike Bublitz < [email protected]> wrote:

...

Dear all,

as it turns out, the cif file was just fine after I had defined the planes properly. I was just accidentally using a script in which I had switched off coordinate refinement. D’Oh. My desk has a bite mark now.

Thanks, Pavel, for spotting this, and thanks to all for your suggestions.

Cheers, Maike

On 28.Feb 2017, at 17:13 , Pavel Afonine wrote:

Excellent point! Have a look at *.geo file that lists absolutely all geometry restraints that are used in refinement (bonds, angles, torsions, planes, chirals, non-bonded, ncs, etc..). If the plane in question is not listed there then we will need to investigate why is that.

Pavel

On 2/28/17 09:01, Tim Gruene wrote:

Dear Maike,

did you read the log file to see whether any of your restraints get acknowledged by phenix.refine? There can be many reasons why they are not, e.g. mismatching atom names, mismatching residue abbreviation, ...

Regards, Tim

On Tuesday 28 February 2017 04:44:43 PM Maike Bublitz wrote:

Dear all,

thank you again for all your suggestions. Here’s what I’ve tried, without success:

1) reduce deviation from planarity restraints to very small value or zero -> ligand still gets distorted in refinement

2) Set all bond angle restraints to ideal values and checked sums of angles in planar rings -> ligand still gets distorted in refinement

3) Used phenix.elbow with - -final-geometry on a pdb of a planar ligand —> ligand still gets distorted in refinement

4) Used the grade server (grade.globalphasing.orghttp://grade.globalphasing.org http://grade.globalphasing.org/) to generate cif file from SMILES string—> phenix.refine stops with message “Unknown file format: grade-ligand.cif”.

Does anyone have another suggestion, except for installing an extra QM package?

Best, Maike

On 27.Feb 2017, at 17:24 , Dale Tronrud mailto:[email protected] > wrote:

You could also check that the other restraints in your cif are compatible with planarity. If the sum of the three bond angles for a planar atom is less than 360 deg your angle restraints will be fighting with your planarity. You also have to check the sum of the internal angles of your planar rings. If I recall correctly the sum of the internal angles must equal n*180 - 360 for the ring to be flat.

Dale Tronrud

On 2/27/2017 9:05 AM, Maike Bublitz wrote: Dear all,

this has probably been discussed many times before, but I just can’t seem to find a solution for my current problem with phenix.refine:

I am trying to refine a protein structure with a rather complex ligand. I’ve generated a .cif file for the ligand with eLBOW, including planarity restraints for its aromatic ring systems. However, phenix.refine keeps on distorting the 6-ring in my indole group, although all respective atoms are included in the plane definition. At a resolution of 3.0 A, I’d very much prefer to impose an ideal planar structure on this ligand.

How can I convince Phenix to not distort my ligand's aromatic rings?

Many thanks for your kind suggestions.

Best regards, Maike

************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk/http://bioch.ox.ac.uk http://bioch.ox.ac.uk/

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.ukhttp://bioch.ox.ac.uk http://bioch.ox.ac.uk/

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.uk

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************************************************* Dr. Maike Bublitz Associate Professor of Microbial Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom phone: +44 (0)1865 613221 <+44%201865%20613221> lab: +44 (0)1865 613318 <+44%201865%20613318> maike.bublitz(at)bioch.ox.ac.uk

If restraint weighting is 1/sigma^2 then setting sigma=0.0 may produce some interesting results. I suspected that setting them to zero might turn the restraints off but can't find a reference to support that. phenix.refine writes a .geo file that lists geometry restraints and sorts them within the file by residual (presumably weighted deviation). That's the first place I'd go look for outliers resulting from your definition of the geometry and compare residuals before/after refinement for what terms are being minimized at the expense of others. Phil Jeffrey Princeton On 2/28/17 11:44 AM, Maike Bublitz wrote:

Dear all,

thank you again for all your suggestions. Here’s what I’ve tried, without success:

1) reduce deviation from planarity restraints to very small value or zero -> ligand still gets distorted in refinement

2) Set all bond angle restraints to ideal values and checked sums of angles in planar rings -> ligand still gets distorted in refinement

3) Used phenix.elbow with - -final-geometry on a pdb of a planar ligand —> ligand still gets distorted in refinement

4) Used the grade server (grade.globalphasing.org http://grade.globalphasing.org) to generate cif file from SMILES string—> phenix.refine stops with message “Unknown file format: grade-ligand.cif”.

Does anyone have another suggestion, except for installing an extra QM package?

Best, Maike