Hi Sitaram,
If you are wondering if the anisotropy is due to cryofreezing, you can
consider the following:
1are you doing a one-step quick soak, slightly longer soak, or serial
soak in increasing amounts of cryoprotectant? There are some
variations to play around with here.
I presume you have detergent in the solution? Are you using the same
detergent for membrane extraction and purification or doing any
detergent exchange prior to crystallization? Have you checked that the
concentrator you use for final protein concentration is okay in terms
of MWCO considering the CMC of the detergent in the solution? The
chances of these affecting the overall diffraction and not just
anisotropy may be higher but still worth considering and trying
alternatives. How about presence of impurities and/or trying
additional purification steps or trying with tags on/off or different
tags (I see you have already tried different constructs)?
Worth putting up a crystal at room temp at taking a single 1 or 2
second exposure (at a synchrotron) to verify the
anisotropy/cryofreezing.
Some other things to consider are better construct optimization
(checking structures of similar targets for construct boundaries,
secondary structure predictions, homology modeling), trying homologs
(including homolog search based on sequence-crystallization propensity
predictions), rescreening with a large HT screen for other
crystallization conditions, hanging drops vs sitting drop
optimizations (not just additives), as well as just trying to solve
your structure from whatever full data set you can currently obtain
even if highly anisotropic.
Thanks,
Debanu.
On Wed, Jul 15, 2015 at 5:09 PM, Sita Ram Meena <[log in to unmask]> wrote:
> Dear all
>
> I wanted to know the suggestion/opinion about anisotropy I am getting in my
> crystal diffraction.
> Things about crystal: one direction goes 2.8A and another is 6 A.
> cell parameters a=133, b=133, c=289, 90, 90, 120, SG=P3
> Protein size: 45 KDa, membrane transporter protein, full size crystal grows
> at 20C in 10days, rod shaped.
>
> Things already tried: 1). additive screen from Hampton (has different 96
> additives), 2).4C crystallization, 3). propylene glycol, 4). controlled
> dehydration, 5).memgold, memgold2, memsys and memstart screens as additive
> in my optimized condition, 6). incubation of crystals plates at 4 C before
> freezing for overnight, 7). crystallized with substrate, 8). pH screen, 9).
> Crosslinking using glutaraldehyde, 10). Seeding, 11). tried many different
> construct (all construct doesn't give crystal as well). 12). tried most of
> conventional cryo.
>
> Crystallization in LCP and with antibody are in consideration.
>
> Now, does some has experience that freezing can cause the anisotropy (I can
> try room temperature, I don't know crystal will survive or not).
>
> Your valuable suggestions are most welcome.
>
> Thanks
>
>
>
> Best
> Sitaram
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