[phenixbb] Few questions about the model building and refinement of cryo-EM data

Pavel Afonine pafonine at lbl.gov
Mon Aug 28 09:51:33 PDT 2017

Hello Bing,

> 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 
can try

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 
> formart)

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?

See above.

> 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:

phenix.models_as_chains model.pdb

> 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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