Hi Ethan, thanks for your reply. The correct situation is the former: hydrogens added at idealized positions *before* refinement and then subjected to refinement along with the rest of the model.
After refinement, MolProbity (the online server) does indeed remove any hydrogens in the PDB file and add them back at idealized positions for the purpose of its calculations, but I am most definitely *not* talking about depositing these post-refinement hydrogens manipulated by MolProbity.
Matthew
---
Matthew J. Whitley, Ph.D.
Research Instructor
Department of Pharmacology & Chemical Biology
University of Pittsburgh School of Medicine
From: Ethan A Merritt
Sent: Thursday, February 27, 2020 6:57 PM
To: Whitley, Matthew J
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Hydrogens in PDB File
On Thursday, 27 February 2020 15:35:05 PST Whitley, Matthew J wrote:
> Hello all,
>
> I am nearly finished refining the structures of two mutant proteins from
> crystals that diffracted to very high resolution, 1 Å and 1.2 Å,
> respectively. Refinement was conducted in the presence of explicit
> hydrogens on the models. I am preparing to deposit these models into the
> PDB but am unsure about whether to retain or remove the hydrogens for
> deposition. On one hand, these hydrogens were explicitly used during
> refinement, so that makes me want to keep them, but on the other hand, they
> were added at theoretical positions by MolProbity’s reduce tool for
> refinement and were not positioned on the basis of experimentally observed
> electron density, so that makes me want to delete them from the
> experimental model. Which is the preferred option for this situation?
The order of operations you describe is unclear.
If you explicitly refined hydrogens then their final positions are indeed
based on experimentally determined data.
The fact that you initially placed them into ideal geometry is not really
any different from the non-H atoms of individual protein residues in your
model, whose original positions were also based on known stereochemistry.
On the other hand, if you mean that the hydrogens you used for refinement
were deleted and replaced during validation by Molprobity (which I think it
may do by default) that's not good. You should rather keep the hydrogen
positions from refinement, not the ones from Molprobity.
Assuming (since this is ccp4bb) you refined with refmac...
- If you are at the level of investigating hydrogen positions, you may want
to consider taking the refinement into shelxl.
- If you are not making claims about hydrogens but just want to describe
what you did during refinement, I'd go with taking them out and settling
for the standard record in the resulting PDB file:
REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
which looks like this in the corresponding mmcif file:
_refine.details 'Hydrogens have been added in their riding positions'
Ethan
>
> Thanks,
> Matthew
>
> ---
> Matthew J. Whitley, Ph.D.
> Research Instructor
> Department of Pharmacology & Chemical Biology
> University of Pittsburgh School of Medicine
>
>
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--
Ethan A Merritt
Biomolecular Structure Center, K-428 Health Sciences Bldg
MS 357742, University of Washington, Seattle 98195-7742
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