[phenixbb] Estimation of ligand binding affinity by occupancy refinement

[Pavel Afonine]

Hi Zoran,

> 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
proteins.
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.

Pavel