[phenixbb] Few questions about the model building and refinement of cryo-EM data
pafonine at lbl.gov
Mon Aug 28 09:51:33 PDT 2017
> General strategies: I select COOT for the model building; using
> phenix.real_sapce_refine for the real space refinement with secondary
> structure restrain and REFMAC for the reciprocal space refinement.
what's the purpose of reciprocal space refinement if you don't have any
measured reflections ? (it's cryo-EM data, which is map!).
> Few questions are listed here.
> 1. The map was too bigger to open it in COOT. The
> phenix.map_to_structure_factors was used to obtaine ~120 MB sized MTZ
> file (still a little big for my computer). I manually build up the
> whole ball-shaped phage with the rigid body fit in COOT (from two
> X-ray structures to 120 chains). My first question will be: In this
> case, should I crop the map in Chimera or other software and only
> focus on a small asymmetric unit to do the model building and the
> followed refinement.
Sometimes box is way larger than actual model. If that's the case you
phenix.map_box model.pdb map.mrc
Also, you can do
phenix.map_box model.pdb map.mrc selection="chain A"
that will give you a box with map and selected part of model.
> 2. I would like to do a real space refinement after the model building.
> Input files:
> A pdb file I just built up from COOT
> A original MAP file
This is all you need for real-space refinement.
> A transfered MTZ file
This is fiction, you don't need it.
> Two restraint files from two X-ray structures by ProSMART (TXT
Phenix does not recognize ProSMART. You can use Phenix tools to generate
secondary structure restraints, such as
> The refinement parameters I would like to select in GUI interface:
> minimization_global, rigid_body, local_grid_search, adp
At 6A resolution you are not going to see sidechains, so no need to do
> Use secondary structure restraints
> Reference model restraints: use starting model as reference, main
> chain, side chain, fix outliers, secondary structure only
> Rotamer restraints
> Ramachandran restraints
> Show per residue
I'd do a default run first to see what happens.
> My second set of questions: Should I select the MAP file (~870 MB) or
> the MTZ file (~120 MB)?
If MTZ file is a FT of the map (full box of reflections, not a sphere)
then both files (map and mtz) contain exact same information, one in
real space and the other in reciprocal space. I'd use original data
(map), not manipulated one (mtz).
> Is that necessary to add the two restraint files from ProSMART if I
> use the starting model as reference?
Phenix does not recognize ProSMART.
> Is the refinement parameters selected properly?
> 3. I gave a try by phenix.real_space_refine. An first error showed up:
> Number of atoms with unknown nonbonded energy type symbols: 6840
> "ATOM 184 HG1 SER 1 12 .*. H "
> "ATOM 458 HG1 SER 1 30 .*. H "
> "ATOM 699 HG1 SER 1 45 .*. H "
> "ATOM 720 HG1 SER 1 47 .*. H "
> "ATOM 762 HG1 SER 1 50 .*. H "
> "ATOM 1241 HG1 SER 1 81 .*. H "
> "ATOM 1465 HG1 SER 1 95 .*. H "
> "ATOM 1747 HG1 SER 1 113 .*. H "
> "ATOM 1758 HG1 SER 1 114 .*. H "
> "ATOM 2173 HG1 SER 1 141 .*. H "
> ... (remaining 6830 not shown)
Looks like PDB file is bad. Serine residue does not have HG1, it should
be HG. Get rid of H by using
phenix.reduce model.pdb -trim > model-no-h.pdb
> I tried phenix.ready_set to fix this problem according to a previous
> discussion but it gave me another error: ENDMDL record missing at end
> of input.
> Thus my third question will be how to fix the first error?
Looks like your file contains several models (MODEL-ENDMDL). This is not
supported. Convert it into multi-chain model:
> Thank you for patience! I would really appreciate your help!
You are welcome! Let us know should you have any more questions or need
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