Very long soaking time will also help. In your question why the binding kinetics was so slow Here are some explanations Diffusion Rate depends from solution viscosity, size of the molecule that moves in this solution and the pores it has to go through. The binding kinetics in the crystals are usually low (compare to solution) Because -pores ligand has to diffuse through solvent channels that sometimes are really narrow (1 molecule only can go through) -size Ligands sometime are big molecules (ions are moving really fast) -viscosity the worst of all the experiment (most of the times) take place in a very viscous environment (high PEG, high salt concentration)
George -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Joern Krausze Sent: Friday, November 28, 2008 4:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Co-crystallization Dear Priya, How long did you soak your crystals? I had a similar problem. When I tried to co-crystallize my protein with its substrate (5 mM) I never got any substrate bound. I tried soaking with higher concentration (up to 50 mM) and this didn't work either. The crystals were infinitely stable in the soaking solution but even after 72 hours there was no substrate bound. When I was almost giving up I tried one last crystal which was in the soaking solution for a long time. I mean a very long, several weeks or so. In this crystal the substrate was bound by the protein. I don't really know why the binding kinetics was so slow. The enzyme's affinity for the substrate was quite high and I didn't expect an unusually long soaking time. Maybe a very long soaking time could also help in your case? Joern From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Priya Mudgal Sent: Thursday, November 27, 2008 11:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co-crystallization Dear All, I am trying to co-crystallize a protein with substrate/substrate analogue and for some reason it is not working. I have tried to co-crystallize with upto 2 mM substrate and it does not bind. The protein precipitates out if I go higher concentration. Soaking does not work either. Is there any suggestion that I should try before I give up on this. Thanks a lot. Priya -- Priya Mudgal PhD Student Duke University ****************************************** Address: Joern Krausze University of Leipzig Centre for Biotechnology and Biomedicine Deutscher Platz 5 04103 Leipzig Germany eMail: [EMAIL PROTECTED] Phone: +49 (0)341 9731312 Fax: +49 (0)341 9731319 ******************************************
