Dear Sue, This is a very interesting case! Normally the Zn-S bond is quite strong - so it's an unusual situation to have. Did you already attempt to purify the protein in the presence of tiny quantities of Zn? Also, what buffers and other components are you using during purification, and what is the chemical composition of the crystallization solution (if you don't mind telling)? Finally, does the purified protein have high activity before crystallization? In other terms - does it have full Zn occupancy before you crystallize it - or does the disulphide form during expression or purification? This would change some of the other solutions that you might attempt.
Cheers, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sue Roberts Sent: Thursday, September 25, 2008 5:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] losing zinc during crystallization Hello Everyone I've been trying to crystallize a zinc-containing enzyme for what seems to me to be an eternity. The protein contains stoichiometric zinc (1 zinc/ protein monomer) when isolated and the zinc is required for activity. Each crystal we've obtained has lost the zinc and contains a disulfide bond between two cysteine residues that should be zinc ligands (based on structures of similar proteins). We've tried crystalizing in the presence of reducing agents, crystallizing with substrate analogs, and supplementing the crystallization drops with zinc with no success (and combinations of these approaches). We've obtained a variety of crystals and determined structures, but none contain any zinc. Attempts to insert zinc into the crystal (zinc + reducting agent or zinc alone) have not been successful. Does anyone have any tricks to suggest that might help? Thanks in advance. Sue Dr. Sue A. Roberts Biochemistry & Molecular Biophysics University of Arizona 520 621 8171 [EMAIL PROTECTED]
