How about chloride? I know, there are negative electrostatics, but I think such is seen sometimes for halides. Or, is it possible that there is a side chain flipped?
Jacob On Tue, May 15, 2012 at 9:51 AM, RHYS GRINTER < [email protected]> wrote: > Dear Community, > > 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 > 1mM EGTA > > The protein was purified by nickel affinity/SEC and dialysed into: > 20mM NaCl > 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, > > Rhys Grinter > PhD Candidate > University of Glasgow > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: [email protected] *******************************************
