Hi Amit,
Based on the original analysis of M. Dayhoff (PAM matrix, Dayhoff
substitution probability, Dayhoff, et. al 1978), introduction of Leu->Met would
be the best choice for production of Se-Met derivatized protein.
It would be best to consider a multiple sequence alignment of your target
protein with proteins of similar sequence, ignore the ones that may have
functional importance (from any literature reports) and ones that may be
present in loops or may be surface exposed (easily done with some prediction
algorithms, would be best to have the Se-Met in well-ordered regions) and then
try multiple combinations of Leu->Met substitutions.
This has been successfully exploited in several cases and a partial list
follows:
Gassner, N. C., Baase, W. A. & Matthews, B. W. (1996). Proc. Natl Acad. Sci.
USA, 93, 12155-12158
Leahy, D. J., Erickson, H. P., Aukhil, I., Joshi, P. & Hendrickson, W. A.
(1994). Proteins, 19, 48-54
Jones, D. T., Taylor, W. R. & Thornton, J. M. (1992). Comput. Appl. Biosci. 8,
275-282.
Good luck!
Debanu.
---
Debanu Das,
JCSG Structure Determination Core,
SSRL, SLAC National Accelerator Laboratory,
Menlo Park, CA.
________________________________
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of amit
sharma
Sent: Tuesday, February 03, 2009 5:02 AM
To: [email protected]
Subject: [ccp4bb] putting in methionines for SeMet crystals
Dear All,
I have a 9-kDa protein that crystallizes well. Since there is no structural
homologue for this molecule, I intend to make Se-Met derivative of the protein.
The molecule has no Met/Cys residues in its sequence. I wanted to know where in
the sequence should I mutate, so that the folding/crystallizability of the
protein is not compromised.
Any suggestions would be of great help.
Thanks in advance,
--
Amit Sharma, Ph.D. Research Associate, Department of Biology,
University of York, YO10 5DD UK
