P.S.:
If your crystals are big enough, try to harvest and wash some and run
on a gel or mass spec to verify that you actually have your target
crystallized and not some other protein as impurity from the prep.
Based on your crystal size you can estimate how many crystals you may
need to harvest for mass spec of SDS-PAGE.
You should also try to get a unit cell from your 7A non-cryo
diffraction if possible.
Best,
Debanu
On Fri, Mar 25, 2016 at 8:31 AM, Debanu Das <[log in to unmask]> wrote:
> Hi Herojit,
>
> Have you tried growing the crystals in cryo? If not, just set up some
> drops in 15-20% GOL, 15-20% EDO, etc. and freeze directly. This should
> take care of crystals being unstable in cryo soaks.
>
> Since you have already tried several things for crystal optimization,
> and if it is a molecular replacement case, you can try collecting ~7A
> data set if you can grow the crystals in cryo, and try solving the
> structure to check for crystal packing, etc. This may give you some
> leads for protein engineering (construct optimization, loop deletion,
> etc.).
>
> You can also try seeding, different crystallization configuration
> (hanging vs sitting), changes in protein purification protocols.
>
> Regards,
> Debanu
> --
> Debanu Das
>
> On Fri, Mar 25, 2016 at 3:01 AM, Herojit khun <[log in to unmask]> wrote:
>>
>> Hi all,
>>
>>
>>
>> Condition of a rod crystal contains 8% PEG 3350, Tris 50mM pH 7.5, 100mM
>> Na2SO4. Buffer (Protein) is tris 50mM, NaCl 50mM, ph 8.5. Most of the
>> crystal diffracts upto 7 A only with Ice rings (without cryoprotectant).
>> Crystals were grown in two weeks.
>>
>>
>>
>> With most of the cryoprotectants including 15% Glycerol(even lower %), the
>> crystals are unstable, starts melting.
>>
>>
>>
>> Additive screening, changing of parameters of condition like pH,
>> temperature, salt concentration did not work. Some crystals grows with 12%
>> PEG 3350, but still it behaves the same.
>>
>>
>>
>> Kindly suggest to improve crystal quality, and cryoprotectant.
>>
>>
>>
>> -- Regards
>>
>> KHUNDRAKPAM HEROJIT
>> NATIONAL INSTITUTE OF IMMUNOLOGY (NII)
>> NEW DELHI, INDIA. 110067
>>
>> NATIONAL INSTITUTE OF IMMUNOLOGY (NII)
>> NEW DELHI, INDIA. 110067
>>
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