As I'm a relatively new to protein crystallography this might turn out to be an obvious question, however.
I'm working on the structure of a enzyme requiring Ca2+ for activity and with calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, in a crystal structure with Ca in the crystallisation conditions (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg).
When Ca is omitted from the crystallizing conditions and a divalent chelator (EGTA) is added the crystals are of significantly lower resolution (3.13A). Refinement of this data reveals density for a molecule coordinated by the Ca coordinating Asp and backbone, however this density is significantly further away (3.4-3.8A) too far away for water or a strongly coordinated divalent cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The density is also much weaker than for Ca in the previous model disappearing at 3.5 sigma.
The crystallisation conditions for the Ca free condition is:
0.1M Tris/Bicine buffer [pH 8.5]
8% PEG 8000
30% Ethylene Glycol
The protein was purified by nickel affinity/SEC and dialysed into:
20mM Tris [pH 8.0]
A colleague suggested that sulphate or phosphate could fit at these distances, but these ions have not been added at any stage of the crystallisation process.
Could anyone give me some insight into what this density might represent?
Thanks in advance,
University of Glasgow