Re: [ccp4bb]: Refinement of ligand occupancy

[Bart Hazes <bhazes@ualberta.ca>]

***  For details on how to be removed from this list visit the  ***
***          CCP4 home page http://www.ccp4.ac.uk         ***

On Wed, 21 Aug 2002, Daniel Copeland wrote:

> ***  For details on how to be removed from this list visit the  ***
> ***          CCP4 home page http://www.ccp4.ac.uk         ***
>
> I am a graduate student working on my first structure determination of a
> heme protein (HOPEFULLY) bound to a ligand.  The assumption is that the
> ligand will displace AN axially bound water molecule.  I have run into a
> problem with trying to refine the occupancy of the ligand.  I have already
> attempted to use CNS to refine the occupancy of the ligand.  I have used
> both the q-group and q-individual and harmonically restrained (with various
> intensities) all or part of the ligand.  I generated another Fo-Fc electron
> density map after the>refinement step and did not see any improvement.  I
> have a few questions and any opinions would be helpful.

As Eleanor already indicated your big problem is to distinguish between
B-factors and occupancy which are highly correlated unless you have very high
resolution. I was surprized to hear though that peak hight was useful to
estimate occupancy since it again is affected by both occupancy and B-factor.

What you have to do is to independently determine either B-factor or
occupancy, then keep that value fixed and refine the other. I think the new
refmac TLS refinement may help you to do just that. Empirical results indicate
that atomic motion can be modeled to a large extend as correlated movement of
groups of atoms. TLS models these correlated motions. If you now define a
group that includes your ligand and atoms in its immediate environment as a
TLS group you will get estimates of the B-factors for your ligand that are
based on all atoms in the TLS groups rather than just the atoms in your
ligand. You can then fix these B-values and refine the occupancy of the
ligand. The beauty is that you can do TLS refinement at limited resolution.

> 1)  Are there other routines that are recommended for refining occupancy of
> a ligand?  For example should the whole ligand be restrained or constrained
> at the beginning of refinement and then the restraints or constraints lifted
> later in refinement and allow the ligand to refine freely?

Yes the whole ligand should be restrained throughout the refinement. Of course
the ligand atomic positions have to be "perfectly" modeled before you start
estimating occupancy otherwise it will be underestimated.

> 2)  What kind of positive and negative indications should I be paying
> attention to - for example should values of refined occupancy or B-factors
> change SUBSTANTIALLY?  What indication would I see if the ligand is PRESENT
> But at low occupancy?  Should I expect to see any changes in the R-factor or
> Rfree values after only 1 cycle of refinement?

If occupancy refines to below zero or significantly above 1 you obviously have
a problem. Whether parameters change SUBSTANTIALLY depends on how close the
starting values are to the refined values, this could be a small or large
change, no way to tell in advance. You must have seen density for your ligand
in order to be able to model it in. That tells you it is there, the occupancy
is another indicator. If the "(HOPEFULLY) bound to a ligand" means you
actually don't see any density and placed you ligand based on homology or
other data then occupancy is your only criterium but it is going to be a low
value and I suggest you don't recommend me as a reviewer of your paper.

Bart

===============================================================================

Assistant Professor
Dept. of Medical Microbiology & Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta, T6G 2H7, Canada
phone:	1-780-492-0042
fax:	1-780-492-7521

===============================================================================