Dear Enrico,
You are right that the trick has its limitations and I am aware of it. However,
it might not be as bad as you think. Fiddling with the crystallization buffer
or transferring crystals to different buffers also causes stress to the
crystals and in many cases loss of resolution. If it turns out that the
reservoir solution freezes ok (or the crystallization drop itself if that is
feasible), I would risk, as I said, trying to freeze a crystal directly from
the drop without any further manipulations. Why try to cryoprotect a crystal
when it is not necessary? If that does not work, I would go for more elaborate
protocols, which, as far as I know, also do not have 100% guarantee of success.
In that case I would also consult the literature like the excellent paper you
mention.
Best,
Herman
-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Enrico
Stura
Gesendet: Dienstag, 27. August 2013 11:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] cryoprotection
Herman,
The trick you suggest is not as valid as you may think. The ice rings can
originate from the crystal itself.
If you crystallize in a high concentration PEG precipitant you will avoid ice
rings, but if you transfer or soak your crystals in the same solution the high
molecular weight PEG will not enter the crystal lattice and you will still get
ice rings.
I have a picture of this in:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.
Crystal Growth & Design, e-print
http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G.
So cryoprotectants need to penetrate the crystal lattice to prevent ice rings,
but even in the presence of ice rings the data can be used.
Regarding optimization:
The main problem you encounter in cryoprotection is that some compounds like
glycerol and ethylene glycol solubilize protein crystals, but if you create a
mixture of various compounds that is precipitation-solubilization neutral, then
there is no real need for optimization.
Enrico.
On Tue, 27 Aug 2013 08:30:28 +0200, wrote:
> A trick I like is just to freeze the reservoir solution or would-be
> cryo-solution without a crystal present. If the frozen solution stays
> clear and does not show ice rings on e.g. a home source, it is worth
> trying. Otherwise, the solution needs optimization.
> Herman
>
>
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
> Uday Kumar
> Gesendet: Freitag, 23. August 2013 19:52
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [ccp4bb] cryoprotection
>
> Hello
>
> Can anyone suggest a cryoprotectant for the following crystallization
> condition
>
> 0.2-0.4M sodium formate
>
> ~20% PEG 3350
>
> 0-25 mM Nickel
>
> 0-100 mM Malonate
>
> Thank you
>
> with regards
> uday
--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
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http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71