Re: [ccp4bb] precipitation of the protein in crystallisation solution

- Protein crystallography

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

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Subject: SLS PX Beamlines: Call for Proposals is Open - Deadline June 15, 2009
From: Stefan Mueller stefan {- dot -} mueller {- at -} PSI {- dot -} CH
Date: 2009-06-03

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Subject: Re: Official PDB Nomenclature?
From: Dirk Kostrewa kostrewa {- at -} LMB {- dot -} UNI-MUENCHEN {- dot -} DE
Date: 2009-06-03

Subject: Re: precipitation of the protein in crystallisation solution
From: Enrico Stura estura {- at -} CEA {- dot -} FR
Date: 2009-06-03

Given that you are able to achieve a protein concentration of 15 mg per ml
in Tris-HCl, pH 8.0, 100mM NaCl and 1% Glycerol suggests that you have no
drastic solubility problem.

Unless you have a chromatophore as an intrinsic co-factor of one of your
component proteins, the yellow slime may be due to impurities.

The "standard" screens in most kits are fine for "standard" proteins but
not for proteins that are out of the ordinary.

The fact that precipitation is absent or delayed with PEG is coherent with
your ability to concentrate the protein to 15mg/ml.

Now for the improvement steps:
The first step is to "normalize" your protein concentration :
## The mean crystallization condition is around 16% PEG 3350. ##
The above may not be entirely correct but is a good working hypothesis
that can be used as described below:
If 16% PEG 3350, 50mM Tris-HCl, pH 8.0, 100mM NaCl and 1% Glycerol gives
you just some precipitation and 24% PEG in the same salt and buffer
conditions
precipitates most of the protein, then you can assume that at 15mg/ml your
protein
concentration is just right and now you may want to screen various ions
and different
pH to find suitable crystallization conditions. (If not ... reduce your
protein concentration to
satisfy the test based on the hypothesis).
The next step is "basic buffer component substitution" to see what your
buffer components are doing:
That implies checking how other salts can be used instead of NaCl (Li2SO4,
KCl Na-mono,di,tri-carboxylates, .... )
1% glycerol replaced by 0.01-5% organic compounds such as MPD, EtOH, PrOH,
xylitol, etc.
Tris-HCl replaced by other basic range buffers such as imidazole, glycine,
... pH 8.5-10.6
If the protein tollerates neutral and acidic conditions you can extend the
pH range to be searched.

Once you understand your protein complex better you should design a screen
of your own based on your
new knowledge.

--
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

CCP4bb navigation

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

Previous message:
Subject: SLS PX Beamlines: Call for Proposals is Open - Deadline June 15, 2009
From: Stefan Mueller stefan {- dot -} mueller {- at -} PSI {- dot -} CH
Date: 2009-06-03

Next message:
Subject: Re: Official PDB Nomenclature?
From: Dirk Kostrewa kostrewa {- at -} LMB {- dot -} UNI-MUENCHEN {- dot -} DE
Date: 2009-06-03