Dear Amit Sharma, you might also consider a SeMet expression of the protein. If it is expressed in E.Coli this may take 2-3 weeks to achieve and save you a lot of hassle compared to a metal soak.
Even if one the the three Mets is at the N-terminus, you could probably get sufficiently strong phase information from Se-SAD or Se-MAD. For phasing on the phosphates you would need very good data and high resolution (certainly better than 2A), but you can of course try. How well do the native crystals diffract? Regards, Tim On Thu, Aug 12, 2010 at 05:09:26PM -0400, amit sharma wrote: > 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 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
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