Hey Joe, set up a coarser grid around the native conditions. I know of one case where semet crystallized nearly 2 full pH units lower than native. A colleague made a couple of years ago a reasonable suggestion: don't do s-met at all, do the crystal screening directly with se-met. Nowadays certainly affordable.
good luck Jens On Tue, 2008-05-27 at 17:11 -0400, Joe Smith wrote: > Dear all, > Sorry for an off-topic query. > I have been unable to crystallize a Se-met containing protein (8 Met > in 206 amino acids) in the native crystallization condition ( 0.1 M > Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). > As expected, solubility of Se-Met containing protein is little less > than the wild type. Other than seeding, i don't know what else I > should try for obtaining a Se-met crystal for phasing. Can I mutate > some of the exposed Met (based on secondary structure prediction and > homologous structure) to Ala as I feel I don't really need 8 Se for > phasing 208 aa long polypeptide. I want to know what generally one > should do when Se-Met containing proteins fail to crystallize. > Thanks in advance. > Joe > PS: Since, protein contains 3 Cys residues.. I am also planning to try > my luck with heavy atom compounds containing Hg.
