Refining the occupancy will help your R-factor and flatten your density,
but you need to be careful to also refine the B-factor of the metal
ion. Don't refine both the occupancy and the B-factor during the same
run (the two are correlated at this resolution), refine the occupancy of
just the metal ion and then refine the B-factor of just the metal ion
(repeat as needed). I used X-plor/CNS to do my refinements, so it was
easy to refine the occupancy (or B-factor) of just the metal ion. After
a few rounds of refinement both parameters will stop changing and you
will have your answer. The final B-factor of the metal ion should be
similar to the amino acid residues that are coordinated to it.
Soaking in several different ion concentrations and collecting
additional datasets is also a good idea (if you have the time). I did
this type of experiment once before (see: JBC 278(14):12271-7. [ Apr 4,
2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845). I soaked in
several different Ca2+ ion concentrations and was able to determine the
binding affinity for that calcium ion using crystallography.
To make sure I was not stuck in a local minima, I would modify either
the occupancy of the B-factor of the metal while keeping the other fixed
and do a refinement. I even tried both large and small changes (both
increases and decreases in value). It always came back to the earlier
answer.
Different Ca2+ ion concentrations can give some additional insight into
the metal binding site. Between the no-Ca2+ structure and the high-Ca2+
structure there was a conserved Asp-residue that changed conformation.
So, I soaked in the appropriate amount of Ca2+ to see the residue in
both positions. There was a high correlation between the asp residue
orientation and the Ca2+ ion occupancy.
Steven Herron
[email protected]
On 5/6/2014 11:02 AM, Chris Fage wrote:
Hi Everyone,
In my 2.5-angstrom structure, there is negative Fo-Fc density
surrounding a metal ion after refining in Phenix. From anomalous
diffraction I am certain of the metal's identity and position in each
monomer. Also, the ion is appropriately coordinated by nearby side
chains. Should I be refining the occupancy of the ion in attempt to
"flatten" the negative density? I am considering soaking the metal ion
into crystals or cocrystallizing and collecting additional datasets.
Thanks for your help!
Regards,
Chris
