[phenixbb] Estimation of ligand binding affinity by occupancy refinement
pafonine at lbl.gov
Fri Apr 27 10:01:26 PDT 2012
> I have a question about sensitivity of occupancy refinement in phenix.
> I have a series of datasets done at our home instrument of relatively
> modest resolutions from 2.8 to 3 A.
occupancies and B-factors are correlated, especial at resolutions around
2.5A and lower; see for example:
Correlation between occupancy and temperature factors of solvent
molecules in crystal structures of
T. Bhat, Acta Cryst. (1989). A45, 145-146.
> These are performed on crystals soaked in progressively higher ligand
> concentrations. We are trying to see to which of the several active
> sites the ligand binds first so we went from low to high ligand
> concentrations. We wanted to estimate the amount of ligand bound by
> refining the occupancies of the ligand. We expect one active site to
> bind more strongly than the other two active sites. This was indeed
> what we observed but when we refine the occupancies of the ligands in
> weaker binding sites starting at 0.2 occupancies they rise to 0.5-0.6
> although some atoms are totally out of density and some are in. B
> factors are reasonable, i.e. don't go wild.
"Totally out of density" is not a precise description. Which level you
use to draw the 2mFo-DFc map? Did you look at 0.5 sigma (that's what I
would use to look at weakly/partially occupied site)? What's the local
map CC? What are the mFo-DFc (both, full and ligand-omit) and 2mFo-DFc
map values at atomic centers of the ligand? phenix.model_vs_data used
with comprehensive=true flag will give you all these values.
Did you refine group occupancy of the ligand - that is one occupancy
factor per whole ligand?
What happens if you run say a few dozens refinements until full
convergence (say 10-20 macro-cycles) each refinement starting with
different initial value of ligand's occupancy and B-factor? Do all
refinements arrive at the same refined occupancy and B? If not, the
spread in refined values will give you the uncertainty.
> My question would be: how reliable is this estimation of ligand
> binding by occupancy refinement?
The test suggested above will give you an idea about the answer.
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