Dear Srinivasan,
The first thing I would do is to look very carefully at the electron
density maps: Does it look like a bunch of water molecules, or is
continuous density present? Could it be some buffer component (Tris,
Hepes, sulfate, DMSO etc) or the counter ion of your ligand or the
cryoprotectant (e.g. glycerol)? Does the protein have disordered
residues e.g. at the N- or C-terminus, which could bind to the binding
site? Same is true for sugars if your protein is glycosylated. If you
are crystallizing a protease, autolysis during crystallization may have
released short peptides which may bind to the ligand binding site. Is
the binding mode of your ligand disordered? Would the electron density
be better explained if you fit the ligand in two alternative
conformations? If your ligand is bound at low occupancy, something else
(e.g. waters, sulfate) may be bound in those binding sites where the
ligand is not present. Also side chains may adopt a different
conformation in the absence of the ligand. In this case one should model
all alternatives.
After this analysis, one usually ends up with a limited number of
alternatives (e.g. ligand is bound, bunch of water molecules, something
else is bound). Most informative is to fit and refine all (in this case
three) possibilities and look which electron density map looks most
convincing. I would also calculate the real space correlation
coefficient for each alternative. If possible, I would also compare the
electron density with the electron density of apo-crystals (not
cocrystallized or soaked with the ligand). If the same density is
present there, it is not your ligand but something else.
Good luck!
Herman
________________________________
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of R.Srinivasan
Sent: Monday, March 11, 2013 11:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] validating ligand density
Hello all,
We co-crystallized an inactive variant of our enzyme
in the presence of substrate and have determined the structure at 1.85A.
Now, we want to validate the fitting of the ligand
into the electron density. We tried validating using the difference map
(2Fo-Fc) after refining the structure without the ligand. But, it is
still a bit inconclusive if the density fits the ligand.
It would be very kind to know if there are tools for
validating this electron density. We were excited about twilight but
turns out it can only be used with deposited structure.
We will appreciate your help and suggestions.
Many thanks,
Srinivasan
