Re: [ccp4bb] putting in methionines for SeMet crystals

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Obtaining relationships between two cross rotation solutions?
From: Francis E Reyes Francis {- dot -} Reyes {- at -} COLORADO {- dot -} EDU
Date: 2009-02-03

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Subject: Re: putting in methionines for SeMet crystals
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2009-02-03

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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of amit sharma
Sent: Tuesday, February 03, 2009 5:02 AM
To: CCP4BB@JISCMAIL.AC.UK
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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Obtaining relationships between two cross rotation solutions?
From: Francis E Reyes Francis {- dot -} Reyes {- at -} COLORADO {- dot -} EDU
Date: 2009-02-03

Next message:
Subject: TeachSG workshop Expertise in Macromolecular Structure Refinement in Prague April 3-4
From: Jan Dohnalek dohnalek {- at -} IMC {- dot -} CAS {- dot -} CZ
Date: 2009-02-03