Another reference with a detailed method for transferring crystals to new conditions prior to soaking is Ray WJ Jr, Bolin JT, Puvathingal JM, Minor W, Liu YW, Muchmore SW. Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by X-ray crystallography at low temperature. Biochemistry. 1991 30 (28),6866-6875. http://pubs.acs.org/doi/abs/10.1021/bi00242a010 and the folloup binding study - Ray WF, Puvathingal JM, Liu Y. Formation of substrate and transition-state analog complexes in crystals of phosphoglucomutase after removing the crystallization salt. Biochemistry 1991 30 (28), 6875-6885. http://pubs.acs.org/doi/abs/10.1021/bi00242a011
Regards, Mitch -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Vellieux Frederic Sent: Friday, May 27, 2011 5:28 AM To: [email protected] Subject: Re: [ccp4bb] need proper suggestion http://scripts.iucr.org/cgi-bin/paper?ms0234 Afshan Begum wrote: > Dear All, > > I have a severe prob lam to performed my ligand binding study with > corresponding protein. I have taking the native diffraction data at > 1.75 A and after that i have performed soaking as well > co-crystallization experiment with my inhibitors. > Problem is that at the active site phosphate ion always bind instead > of inhibitors. I have used 1.6 M ammonium phosphate conc at the > crystallization recipe which is a very weak inhibitor of my protein > whereas the ligand is already clinically applicable but due to the > very high conc. of phosphate i have not achieved my target. If some > one can suggest me what else i can replace with ammonium phosphate or > any other suggestions would be appreciated. > > I have tried to grown crystals some other condition but the crystal > was not diffracted beyond 3.5 A. > > Best Regards > > AFSHAN > I think you could get some hints from the following publication, J. Appl. Cryst. (1988) 21, 426. The transfer of protein crystals from their original mother liquor to a solution with a completely different precipitant, Schreuder H et al. http://scripts.iucr.org/cgi-bin/paper?ms0234 The aim would then be to (try to) transfer your crystals to another mother liquor that does not contain any phosphate. HTH, Fred.
