Dipankar,
What you describe occurs frequently and some times improves with refinement and other times does not. I suggest not adding ligands until the rest of the structure is well behaved - usually around 25-28% Rfree. This will tend to favor the highest quality electron density and will not have bias from the ligand in the phases. Small proteins with electron rich ligands may need the ligand fitted to cross even 30%, but it is a guideline.
Also keep in mind that organic molecules can show true electron density shifts based on the chemistry present. A sulfonamide on a phenyl ring will for instance pull most of the electrons (on average) from the ring, leaving it looking more like a comma than a disc...even at quite high resolution...it is "real" electronics observed visually.
Your second question regarding ~100A^2 for the B-factor of the ligand requires further clarification:
1. Is the resolution of your data sufficient to warrant individual B refinement? If so, what is the average B of residues near the ligand? In general, I ligand at roughly 100% occupancy should have B-factors within 10-20% of the value of the protein. Some programs do a poor job of refining B-factors of ligands. X-PLOR, for instance used to have a hard time with them.
2. What is the relative quality of the electron density of the ligand vs protein? Does it suggest less than full occupancy of the ligand in the pocket? If so, the approximate occupancy of the compound can be estimated by the comparison of the ligand B and nearby protein B factors. A ligand B twice the value of the protein residues would roughly suggest an occupancy of 50%. You can try that and see if the Bfactors stabilize.
Cheers,
Jim
On Wed, Apr 11, 2012 at 2:11 AM, Dipankar Manna
<[log in to unmask]> wrote:
Dear Crystallographers,
The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular
ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing >100. How can I justify this issue. Asking for suggestions.
Regards,
Dipankar Manna
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