Hi Rafael,
If you really want to diffract your crystals frozen, I have two more
suggestions for cryo-procedures which can be tried:
a) annealing
e.g.
Harp, J.; Timm, D. & Bunick, G. Macromolecular crystal annealing:
overcoming increased mosaicity associated with cryocrystallography.
Acta Crystallogr D Biol Crystallogr, 1998, 54 ( Pt 4), 622-8
Yeh, J. & Hol, W. A flash-annealing technique to improve diffraction
limits and lower mosaicity in crystals of glycerol kinase. Acta
Crystallogr D Biol Crystallogr, 1998, 54 ( Pt 3), 479-80
Kriminski, S.; Caylor, C.; Nonato, M.; Finkelstein, K. & Thorne, R.
Flash-cooling and annealing of protein crystals. Acta Crystallogr D
Biol Crystallogr, 2002, 58, 459-71
b) slow cooling
Warkentin, M. & Thorne, R. E. Slow cooling of protein crystals. Physics
Department, Cornell University, Ithaca, NY 14853, USA., J Appl
Crystallogr, 2009, 42, 944-952
Good luck!
christian
Natalie Zhao wrote:
> -----Original Message-----
> From: [log in to unmask] [mailto:[log in to unmask]] On Behalf Of Rafael Couñago
> Sent: 14 December 2009 20:22
> To: [log in to unmask]
> Subject: [ccp4]: TDS upon flashcooling
>
> Dear all,
>
> I got these beautiful looking crystals that grow in high salt (1.8M) and
> diffract under 2.0A at room temp. My attempts so far to cryo protect
> them have resulted in a loss of resolution (2.5A tops) and increased
> anisotropy.
>
> I have tried some of the usual suspects; no cryo, ethylene glycol,
> glycerol (even 5% makes my crystal crack), sucrose, glucose, paratone-n
> (no diffraction at all). I have tried both dipping the crystal straight
> into liquid nitrogen and flash cooling it in the cryostream.
>
> An interesting observation is that the diffraction pattern following
> freezing has a substantial amount of thermal diffuse scattering (but no
> ice rings). If I remove the crystal from the cryostream and re-anneal
> it at room temp (in air or in mother liquor or mother liquor + cryo)
> most of the TDS goes away, but the max resolution is still around 2.5A
> and the higher anisotropy is still there. Extending re-annealing times
> lead to cracking of the crystal.
>
> My two questions would be:
>
> - any thoughts on cryo solutions?
> - does the result from the re-annealing experiment ring any bells?
> Would this be an indication that I need the cooling to be faster or slower?
>
> Cheers,
>
> Rafael.
>
_______________________________________________________________________
Dr. Christian Biertümpfel
Laboratory of Molecular Biology
NIDDK/National Institutes of Health phone: +1 301 402 4647
9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201
Bethesda, MD 20892-0580
USA
_______________________________________________________________________
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