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: owner-c...@dl.ac.uk [mailto:owner-c...@dl.ac.uk] On Behalf Of Rafael > Couñago > Sent: 14 December 2009 20:22 > To: c...@ccp4.ac.uk > 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 _______________________________________________________________________
