Hi Mary,
What final percent MPD do you have prior to flashcooling? Karolin Luger (and perhaps others) found
that the final percent MPD had a significant effect on crystal mosaicity and final diffraction
limit for nucleosome crystals.
For example, if the crystals are left in mother liquor containing 15% MPD, that is suboptimal. On
the other hand, if the MPD concentration was increased beyond 24%, the the crystal mosacity
increased and the diffraction worsened. We stuck to 24% MPD. Did you try playing with the MPD
concentrations? Also, try supplementing MPD with a small percent of sugars like sucrose, trehalose.
Another note: Nucleosome crystals are prone to temperature-dependent lattice changes (always along
c-axis) with a concomitant worsening of diffraction. Even if it sounds anectodal, mythological or
voodoo-ish, we found that it greatly helped diffraction anisotropy and mosaicity if we dunked the
crystals in liquid propane first. After the initial dunking, it was just fine to freeze the
crystals in liq. N2 for longer storage and/or transport. It was the first dunk that was critical.
Good luck!
Raji
---------Included Message----------
>Help please!
>
>I'm looking for some new ideas. I have crystals that come out of a
>sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium
>acetate and MPD for the well solution. The MPD concentration is
>sufficient to act as a cryoprotectant. Currently, I directly freeze
>these crystals in liquid nitrogen. When I collect data, I typically
>have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is
>further complicated with a weakly diffracting crystal (4-5 A) that has
>a long unit cell axis of ~500 and often twinning.
>
>It has been suggested to me that the cryoprotectent is a problem. I
>haven't checked the diffraction at room temperature, yet. Please no
>suggestions of finding a different crystal form as that's not a
>consideration at the moment. I have my reasons. I did find one
>crystal that has lower mosaicity (0.5 to 0.8) but had weaker
>diffraction then the typical crystal. Attempts at flash cryoannealing
>have not helped.
>
>So, what's a good way to change the cryoprotectant if the
>cryoprotectant is the precipitant? I've considered trying dehydration
>but wasn't certain if that would help with the mosaicity.
>
>Thanks for any ideas,
>
>Mary X. Fitzgerald
>Postdoctoral Associate
>
>
---------End of Included Message----------
|