[ccp4bb] Cryo-protectant

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Peptide on two-fold axis - was: PEG molecule crossing a two-fold crystallographic symmetry axis
From: Kris Tesh Kris {- dot -} Tesh {- at -} RIGAKU {- dot -} COM
Date: 2009-05-01

Next message:
Subject: Re: Hanging vs. Sitting
From: Joe gchen2 {- at -} GMAIL {- dot -} COM
Date: 2009-05-01

Subject: Cryo-protectant
From: Marcus Winter Marcus {- dot -} Winter {- at -} VARIANINC {- dot -} COM
Date: 2009-05-01

Further to the other contributions to this discussion, please
note that the Oxford Diffraction PX Scanner system - for diffraction
quality assessment of crystals in situ in the crystallisation plate,
can also play a powerful role in all of this. Thus, in addition to
differentiating salt from protein crystals and non-invasively
identifying the 'best diffracting' crystal in a plate, by in situ
diffraction examination of a crystal before and after the addition
of cryo-protectant to the droplet, then any problems actually
created by the cryo- can be identified.

Thanks.

Marcus Winter (Oxford Diffraction)

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Andy Torelli
Sent: 24 April 2009 16:46
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cryo-protectant

Hi Liew,

There have already been some very good suggestions. I agree
with Tim
Gruene that a great starting point is to test the diffraction properties

of your crystal at room temperature. This can serve as a baseline for
comparing/evaluating cryoprotecting agents and methods.

You can also test your potential cryoprotection solutions to see
if
they freeze clear or even take a few X-ray snapshots of them to confirm
there are no ice rings.

There are lots of publications that can be helpful. One very
helpful
reference is:
Garman, E.F. and Doublie, S.
Cryocooling of Macromolecular Crystals: Optimisation Methods.
Methods in Enzymology (2003) 368, 188-216.

Be aware that, as mentioned previously, the method for freezing
can
make a difference (e.g. freezing in cold-stream vs. plunging in
nitrogen). An obvious difference is different cooling rates (you can
find references for this) or less obvious reasons, for example
differences in dehydration that occur during longer/shorter transfer
through air from drop to cold-source for either method.

Finally, you can check out the database for cryoprotecting
solutions:
http://idb.exst.jaxa.jp/db_data/protein/search-e.php

Good luck,
-Andy

--

=============================================
Andrew T. Torelli Ph.D.
Postdoctoral Associate
Laboratory of Steven E. Ealick
Department of Chemistry and Chemical Biology
Cornell University
=============================================

On 4/24/2009 10:44 AM, Liew Chong Wai wrote:
> Hi all
>
> Thanks for your precious suggestions and ideas.
> The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M
> MgCl.6H2O, 35% PEG3350
> Now, my crystal seem ok in 20% ethylene glycol, but only after a
couple
> minutes of dehydration at room temperature. For sure, i will try other

> cryoprotectant that was suggested here.
> I just wondering why MPD kills the crystal.
> Many thanks
>
> *LIEW*
>
>
>
>
>
>
>

--

=============================================
Andrew T. Torelli Ph.D.
Postdoctoral Associate
Laboratory of Steven E. Ealick
Department of Chemistry and Chemical Biology
Cornell University
=============================================

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Next message:
Subject: Re: Hanging vs. Sitting
From: Joe gchen2 {- at -} GMAIL {- dot -} COM
Date: 2009-05-01