Re: [ccp4bb] how to improve resolution

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

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Subject: Postdoctoral Position in Protein-DNA Crystallography
From: John McGeehan John {- dot -} McGeehan {- at -} PORT {- dot -} AC {- dot -} UK
Date: 2010-02-05

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Subject: Re: how to improve resolution
From: Annie Hassell annie {- dot -} m {- dot -} hassell {- at -} GSK {- dot -} COM
Date: 2010-02-05

Rui--

It is not uncommon to see "hollowed ends" on crystals when they grow
quickly at that point. You have received some very good suggestions thus
far, and slowing down the growth might also be helpful. We have had good
success doing this by just varying the protein concentration and/or the
precipitant concentrations. We have also had good luck substituting
sodium malonate for the ammonium sulfate in our optimizations.

HTH!
annie

Annie Hassell
Glaxo Smithkline
5 Moore Drive
RTP, NC 27709
919/483-3228
919/483-0368 (FAX)
annie.m.hassell@gsk.com

"rui"
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05-Feb-2010 09:48
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Re: [ccp4bb] how to improve resolution

Hi, Thanks all for the good suggestions, I just attached two images that
show my crystals, maybe you'll see new problems with the conditions.
Thanks again.

On Fri, Feb 5, 2010 at 7:43 AM, Enrico Stura wrote:
On Fri, 05 Feb 2010 05:39:14 +0100, rui wrote:

Hi, All,

We are trying to crystallize a protein and found some initial hit in the
following conditions,

pH 4.8, 0.2 M AS or some other salts ( NaCl,LiCl, MgCl2 ), 32% PEG4000 or
PEG3350 ). However the quality of the crystal is not so great,some of them
look like needle cluster(very long in length), some of them look like
multi-crystals or hollow inside.
Such growth problems are likely due to the quality of the protein
solution.
Changes in precipitant concentrations are likely to be ineffective. Try
ion exchange purification.

We tried to optimize the pH and PEG and
tested one that diffracts at 2.9A. For the next, how to improve
resolution?Any suggestions? Even mutate the protein to get a high
resolution
is ok, generally what kind of mutation would make proteins crystallize
better? Thanks.
It is not mutations that will improve diffraction, it is small changes
in crystal contacts.
The Discussion section from:
http://pubs.acs.org/cgi-bin/article.cgi/cgdefu/2007/7/i11/html/cg700698d.html

may help.

Enrico

--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette
Cedex FRANCE http://www-dsv.cea.fr/en/ibitecs/82
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: estura@cea.fr Fax: 33 (0)1 69 08 90 71
[attachment "Fig1.JPG" deleted by Annie M Hassell/PharmRD/GSK] [attachment
"Fig2.JPG" deleted by Annie M Hassell/PharmRD/GSK]

CCP4bb navigation

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

Previous message:
Subject: Postdoctoral Position in Protein-DNA Crystallography
From: John McGeehan John {- dot -} McGeehan {- at -} PORT {- dot -} AC {- dot -} UK
Date: 2010-02-05

Next message:
Subject: SOLVE help
From: Ankit Gupta ankitgpta {- at -} GMAIL {- dot -} COM
Date: 2010-02-05