On Aug 11, 2009, at 11:37 AM, [email protected] wrote:
Is there a chance of backsoaking even when the protein has been expressed as a selenomethionine derivative? I just overexpressed the protein by shutting down the methionine biosynthesis in E.coli and incorporated SeMet instead of methionine. The protein has three methionine and is 26kDa
You could mass-spec the SeMet protein to make sure the expression worked, but these protocols rarely fail. Assuming the incorporation of SeMet is nearly complete, you'd see a huge fluorescence peak even if the methionine sidechains were disordered. Especially if the crystals diffract weakly and your overall signal-to-noise ratio is relatively low, it's not terribly surprising that you don't have much anomalous signal. I've seen lots of data like this (sometimes with many more Met residues); unfortunately, I've never found any solution other than growing better crystals. It might help to collect more frames to improve the signal, but this is also going to increase the radiation damage. -------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]