Hi vito, I don't think you need double labelling. Two (if there is a Met at the N-term it might be less ordered) or three SeMet should suffice to phase 360 residues. It was true in the old days that a Se could phase less than a hundred residues, but if your measurement is good (resolution better than 3A, 360° rotation range, fine slicing, PAD, little radiation damage, stable beamline) then I see no reason why SeMet-SAD (or even better MAD) shouldn't work.
One should mine the literature to find how many residues can be phased with one SeMet nowadays - sorry, lost track. good luck, Kay On Wed, 21 Jun 2017 17:46:56 +0200, Vito Calderone <calder...@cerm.unifi.it> wrote: >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
