Try refining with: Na, Ca or Water at that position and compare the resulting maps. That should provide you with the information you need.
It could be a weakly bound sodium, or calcium ion. It could be that calcium was not fully removed by EGTA treatment. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Tue, May 15, 2012 at 10:51 AM, RHYS GRINTER <r.grinte...@research.gla.ac.uk> 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
