[ccp4bb]: Summary of replies to "Se-Met (again)".

["Gerry McDermott" <gmcdermott@lbl.gov>]

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Hi,

Many thanks to the people who responded to my question about poorly diffracting Se-Met crystals.  I've summarized the question, and the replies below. 

Cheers,

Gerry

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Original question:  

"Native crystals diffract to ~ 2Ang.  Se-Met derivatives of the same only diffract to ~7 Ang at best.   We have tried cryocooling the crystals straight from the mother liquor (it contains enough PEG to allow that), and transferring the crystals to cryoprotectants containing DTT, etc., in the vain hope that this somehow magically improves the resolution.  Advice/suggestions please!


Answers:

Dina Fotinu, Glasgow.
Suggested using a new (to me) cryoprotectant called 'Panjelly' [See: http://www.jenabioscience.com/panjelly.html and J.Appl. Cryst. (1999) vol.32, 1003-1005] and trying annealing as a means of improving the resolution.

Gwyndaf Evans, MRC, Cambridge.
Suggested the alternate approach; checking the inherent order in the crystal by trying to collect some room temperature data - it could be the lack of resolution is simply a result of cryocooling.

Michael Forstner, Uppsala.
Wondered if Se oxidation of a Met crystal contact was effecting packing. He also,  pointed out, that there are additional reducing agents in the Se-Met crystallization drop, that are not present in the native crystallization conditions.  This could be the problem.

Peter Adams, Yale.
Suggested a paper describing one approach to increasing resolution - JMB vol 295 pg 541-556

Hongmin Li, NIST
Recommended working in a nitrogen box to prevent oxidation.

Savvas N. Savvides, Orgegon.
Suggested that dissolving the crystals and re-crystallizing the protein might eliminate a contaminant - denatured protein in the crystallization drop? - and lead to better diffraction


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Gerry McDermott
Macromolecular Crystallography Facility
Advanced Light Source
Berkeley, CA 94720

Tel: (510) 495 2411