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Re: [ccp4bb]: stabilizing SeMet crystals

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We experienced similar disparity between the behavior of Native and
Se-Met crystals for a protein where most of the methionines are exposed
to solvent. 

i) The addition of 50mM mercaptoethylamine during purification and
crystallization helped minimize 'aggregation' problems, and helped
stabilize the crystals.

ii) The use of sitting drop, rather than hanging drop is perhaps a
better option for 'delicate' crystals. Why? The feeling that a drop on a
'flipped over' coverslip is much more likely to evaporate faster - and
hence cause osmotic shock to the crystals - than a drop in a 'sitting
drop' experiment after the sealing tape has been cut open. 

Best of luck,

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


David E. Timm wrote:
> We are looking for suggestions to try improving the stability of SeMet
> substituted crystals.  The native crystals are stable to handling,
> cryocooling, etc.  However, the SeMet substituted crystals are very fragile
> and frequently crack when the coverslip is removed from the tray, when the
> crystal is placed on a loop or when placed in cryo protectant solution.  Out
> of about 50 crystals we have mananged to collect a 2.0 ang data set in house
> using one of the SeMet substituted crystals, which shows no significant cell
> or space group changes compared to the native crystals.  While we have kept
> this crystal in cryogenic storage, it would be nice to have some backups.
> Any suggestions?