Hi Rhis, It may have been suggested already but still... X-ray fluorescence spectra can often tell you what elements you may be dealing with. Spectra won't tell anything about binding specifics, but any extra bit of information could help. Any descent synchrotron MX beamline, equipped for MAD/SAD phasing, should be able to help. Of course, spectroscopy beamlines would be even better. Cheers, N.
Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER Sent: Tuesday, May 15, 2012 9:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange Density 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
