[phenixbb] phenix.xtriage says that entire res range contains anomalous signal but chart says differently?

Nathaniel Echols NEchols at lbl.gov
Tue Aug 11 12:06:55 PDT 2009


On Aug 11, 2009, at 11:37 AM, sbiswas2 at ncsu.edu 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
NEchols at lbl.gov








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