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Re: [ccp4bb] Crystals grown from high ammonium sulphate
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
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Subject: Re: Crystals grown from high ammonium sulphate
From: "Mark J {- dot -} van Raaij" mark {- dot -} vanraaij {- at -} USC {- dot -} ES
Date: 2008-11-04

also, don't forget to measure at least one crystal at RT, probably
most easily done in a Mitegen loop-and-sleeve - to have an idea of the
intrinsic diffraction quality of the crystals without freezing.
Mark
Quoting Savvas Savvides :

> Dear Sabine,
> I recently dealt with a very similar situation as follows:
>
> -I ended up growing the crystals in 4+4 uL drops. Skin formation tends to be
> less of a problem in larger drops. This kind of experimentation is of course
> only possible if protein production is not a limiting factor.
> -For crystal manipulation, I used to add 10-20 uL of the reservoir solution
> directly to the drop. In this way I could easily manipulate the 5-6 crystals
> that grew per drop within 5 minutes without any noticeable effects on the
> crystals.
> -I found out that cryo-cooling the crystals by plunging them into liquid
> nitrogen straight out the drop was the only way to effectively cryo-cool
> such crystals. In fact the crystal condition was very similar to yours (3.2
> M AS, 20 mM potassium phosphate pH 6.0).
>
> I cite the most relevant paragraph from our paper (Kyndt et al.
> Biochemistry. 2007 46(1):95-105.):
>
> "To prepare crystals for data collection under cryogenic
> conditions (100 K), crystals were flash-cooled by plunging
> them directly from their native drops into liquid nitrogen. A
> series of cryocooling conditions using a variety of cryoprotecting
> reagents such as glycerol, sucrose, PEG 400, and
> paratone indicated that only crystals flash-cooled by plunging
> them directly from their native drops into liquid nitrogen
> produced diffraction of acceptable quality."
>
> Best wishes
> Savvas
>
>
> ----
> Savvas Savvides
> L-ProBE, Unit for Structural Biology
> Ghent University
> K.L. Ledeganckstraat 35
> 9000 Ghent, BELGIUM
> office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
> Email: savvas.savvides@ugent.be
> http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sabine
> Schneider
> Sent: Tuesday, November 04, 2008 7:34 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Crystals grown from high ammonium sulphate
>
> Hi everyone,
>
> We got crystals that grew in ~3.2M ammonium sulphate and some
> tris-buffer at 18dgC. Unfortunately the crystals take a while to grow
> (~4-5 weeks) and so far we only have 4-5 xtals.
> I tried to freeze the crystals, but as soon as I broke though the skin
> of the drop the ammonium sulphate started to crystallise. I got the
> crystals out, froze them using sort of an artifical mother liquor with
> sodium malonate as cryo and tested the diffraction. The freezing seems
> to be OK and it is definitely a protein crystal. The crystal suffered
> when the ammonium sulphate in the drop started to crystallise, but
> didn't seem to deteriorate anymore in the cryo. Well the corners had
> already more or less disappeared by the time I got them out of the drop...
> Since we only have a few xtals at the moment and I can't try out a lot
> of things, I was wondering if anyone has experienced and solved a
> similar problem? My freezing attempt so far was in an airconditioned
> room with 18dgC. I thought about higher humidity and temperature in the
> room, and/or adding the cryo directly to the drop....
> Any ideas are very much appreciated!
>
> Sabine
>
> ------------------------------------------
> Dr. Sabine Schneider
> Ludwig-Maximilians-University
> Department of Chemistry and Pharmacy
> Butenandtstrasse 5-13, Building F
> 81377 Munich
> Germany
> Phone: +49 (0)89 2180 77846
> Fax: +49 (0)89 2180 77756
> http://www.carellgroup.de/
>
>
>
>
>
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