Mary,
freezing habitually increases mosaicity. In your case, the high water
content adds to the problem.
Try not to freeze the crystal but collect at sub-zero temperature (in
short glass capillaries or use oil plugs instead).
You have to optimize the "close to freezing" data-collection temperature.
I collected complete synchrotron datasets (of GCPII in buffer with
PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values
of as small as 0.07 degrees! At 277 K, the crystals only last for a few
images and freezing did not work (for the buffer mentioned before).
- J. -
> -----Original Message-----
> From: Mary Fitzgerald <[log in to unmask]>
> Date: Mon, 9 Jul 2007 18:05:10
> To:[log in to unmask]
> Subject: [ccp4bb] Help with reducing crystal mosaicity
>
> 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
>
--
Jeroen Raymundus Mesters, Ph.D.
Institut fuer Biochemie, Universitaet zu Luebeck
Zentrum fuer Medizinische Struktur und Zellbiologie
Ratzeburger Allee 160, D-23538 Luebeck
Tel: +49-451-5004070, Fax: +49-451-5004068
E-mail: [log in to unmask]
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.opticryst.org
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