I forgot to add: have you tried growing your crystals in presence if different cryoprotectants?
Regards,
Debanu
> On Jul 15, 2015, at 5:49 PM, Debanu <[log in to unmask]> wrote:
>
> 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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