Absolutely. But don't bother with SIRAS: straight SAD will do the job 9/10 times these days, thanks to magnificent beamlines and algorithms.
Sent from tiny silly touch screen ________________________________ From: "Whitley, Matthew J" <mjw...@pitt.edu> Sent: 22 Jun 2017 1:09 am To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling I second (or third) the suggestion that others have given to try soaking mercury compounds into your crystal. Mercury absolutely loves free cysteines, and if you have 5, you have a great chance of getting binding. If isomorphism is maintained after soaking, the isomorphous differences will be huge, and if you can collect data at a synchrotron, then the anomalous signal also opens up the possibility of SIRAS phasing. Everybody seems to have their own favorite mercury compound to recommend, and mine is potassium tetraiodomercurate (K2HgI4). Just recently I was able to get mercury binding to three free cysteines in my protein with this compound with absolutely no effort whatsoever. I simply dissolved a few grains of compound in an acetonitrile/water solution, added a drop to my crystal drop, waited 30 minutes, and then collected the data. SHELX was able to locate the sites and solve the phases by SIRAS in mere moments. It couldn't have been easier, and I thank the experimental phasing gods for their generosity. In any event, your protein has a large number of free cysteines, so I think you probably have an above average chance for some flavor of phasing based on mercury to be successful. Good luck! Matthew --- Matthew J. Whitley, Ph.D. Research Associate Department of Structural Biology University of Pittsburgh School of Medicine ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of CCP4BB automatic digest system <lists...@jiscmail.ac.uk> Sent: Wednesday, June 21, 2017 7:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: CCP4BB Digest - 20 Jun 2017 to 21 Jun 2017 (#2017-172) ------------------------------ Date: Wed, 21 Jun 2017 17:46:56 +0200 From: Vito Calderone <calder...@cerm.unifi.it> Subject: Se-Met and Se-Cys double labelling I am working on a protein having 360 residues. In its sequence there are 3 Met and 5 free Cys. I will need MAD to solve the structure since based on the sequence the closest homologue has 20% identity…I suppose MR would be very unlikely to work…so I would like to express a selenium derivative to exploit MAD. Looking in the literature 1 Se-Met every 120 residues seems not to comply the threshold to get a good anomalous signal. For this reason I would like to exploit both Met and Cys so I would have 8 seleniums per 360 residues. Could somenone suggest a reference to a protocol to express the double mutant protein in NON auxotrophic strains of E. coli which you have experienced working efficiently? Thanks
