Hi PhenixBB. 

I have a very minor problem with partial occupancy waters moving out of density during refinement

I have 2 structures, one apo structure (~1.3Å), one ligand bound (~1.5Å).

The ligand binding site contains a Calcium ion. In the apo form there are 2 waters co-ordinating this Calcium. In the ligand bound form, these waters are replaced by oxygen atoms within the ligand. 

However, the ligand is only at 0.5 Occupancy as part of the ligand lies on a crystallographic two-fold axis. The ligand itself refines well with occupancy fixed at 0.5, and the Bs are sensible. 

In the 50% of the molecules which don't co-ordinate ligand, two waters must co-ordinate the calcium ion, and in keeping with this, we see that the electron density around these positions is greater than would be expected for the 50% occupancy ligand. So I added 2 0.5 occupancy waters to the model at these positions. 

However, when I run phenix.refine (refining XYZ, real space, occupancy, and individual Bs (isotropic for the ligand & waters - for the protein I use anisotropic Bs) these two 'ghost waters' move out of the electron density and their Bs increase massively.

My questions are:

Is there any way to restrain these waters to their apo positions? Is this the correct approach?

I had initially thought that reference model restraints might be useful here, but if I understand correctly, reference model restraints cannot be applied to waters - Is this correct?

If I cannot restrain the waters to the apo positions, would I be justified in fixing (constraining) them? This seems a little extreme and I am loath to do it, but given the isomorphous apo structure, I do have evidence that in the absence of ligand waters do take up these positions. 

Can anyone help?

Cheers,

Dave

		David Briggs about.me/david_briggs

 

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