[phenixbb] difference map peaks

Joe Krahn krahn at niehs.nih.gov
Tue Oct 6 16:37:11 PDT 2009

> Hi everyone,
> I would like to have commnets from experts on the following issues.
> I am refining DNA-protein complex at 3 A resolution and my current Rfree 
> is 21.5 % (phenix.refine).
> 1. 
> I am getting difference map peaks (in Coot) of magnitude less than -5 
> sigmas (0.24 e/A3) (~10 or more peaks) and equal number of positive 
> peaks of same magnitudes.
> The positive peaks hardly have 2fo-fc of more than 1.0 sigma level. I 
> presumed this well could be due to bulk solvent or improper mask. 
> Therefore I optimized the mask parameters (by giving option under 
> "General refinement parameters" in phenix.refine GUI). I could get my R 
> free lower as expected but I still got these peaks back. Although I am 
> not absolutely sure, but positive peaks are more in  polar pocket of 
> protein and negative peaks are more in non-polar and aromatic pockets. I 
> have PEG, Na acetate in my crystal soup. What these negative peaks 
> represent for?
I suspect that negative peaks are from the bulk-solvent mask, as you 
suspected. Some proteins have hydrophobic cavities that are big enough 
to make a small void that ends up with a bit of bulk solvent density. 
Optimizing bulk solvent parameters does not necessarily help, because 
similar sized cavities may in fact have a bit of actual solvent density.

It may be possible to put zero-occupancy dummy atoms on those sites to 
screen out the bulk solvent mask, but I don't know how the PHENIX bulk 
mask handles partial occupancy.
> 2. 
> During addition of atoms like Na+, Cl- in map, do one need to careful 
> about of the its coordination valency in surrounding pocket. ?
Definitely. The PDB is full of misidentified ions, even in active sites 
at resolutions much better than 3.0. Many Polymerase Beta structures 
have an Mg++ ion in the model, where the true identity is Na+. It is 
easy for ions to have partial occupancy with waters or other ions, or 
some disorder due to the water ligands.

Of course, there are probably even more invalid waters in the PDB than 
anything else. My opinion is that it is better to make your best guess 
rather than just leave in phony waters, and put remarks to the deposited 
PDB file for anything that is uncertain. Unfortunately, most PDB 
consumers don't read remarks, so we would benefit from a standard way to 
flag qualitative, author-defined uncertainties.

Joe Krahn

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