Hi Amit, For heavy atom phasing, you'll have to try all the things you can, limited only by supply of crystals, availability of heavy atoms (and considering heavy atom handling and waste disposal policies for solutions, tips, etc.) and time and effort.
I had success with both short soaks (tried from 5-120 mins) at higher/medium (1-10 mM, depending on compound solubility, etc) concentration and long soaks (days > 1) with lower concentration. Take a look at the following paper which is one of the first articles describing in detail short soaks: Acta Cryst. (2002). D58, 1092-1098 Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases P. D. Sun, S. Radaev and M. Kattah In you r case, brominated DNA would also be a good option without doing soaking. I think there is a paper also on phasing of the P in the DNA backbone. Best, -Debanu. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of amit sharma Sent: Thursday, August 12, 2010 2:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] heavy atom soak Dear All, In order to phase, I intend to derivatize my protein(30kDa)-DNA(7.2kDa) complex with heavy atoms. I wanted to know which was the better way to do it: longer soaks at lower heavy atom concentration, or shorter soaks with higher concentration of heavy atom. Also, what concentrations and time is generally used in either case. The crystals came up in pH 6.0 buffer and the protein contains 1Cys, 3Met and 3His residues. I would appreciate any advice or link to the literature. Many thanks in advance Amit Sharma, Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University, Baltimore, MD21218
