Re: [ccp4bb] crystallization tips for acidic proteins

- Protein crystallography

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

Previous message:
Subject: FW: .getTwoTheta() in DiffractionImage library
From: "Remacle, F (Francois)" f {- dot -} remacle {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-07-02

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From: Sergiy Klyuyev klyuyev {- at -} UAB {- dot -} EDU
Date: 2008-07-03

Subject: Re: crystallization tips for acidic proteins
From: Tom Huxford thuxford {- at -} SCIENCES {- dot -} SDSU {- dot -} EDU
Date: 2008-07-02

An easy (and inexpensive) screen for crystallization of highly charged
proteins is the phosphate screen. It just takes two stock solutions (5
M NaH2PO4 and 3 M K2HPO4) and water. Start with a two-dimensional
screen where the the total phosphate concentration varies from about
0.4 M to 2.0 M in one dimension and the ratio of monobasic to dibasic
phosphate varies (1000:1, 100:1, 10:1, 1:1, etc.). After you make
initial observations of solubility then refine with finer and finer
ranges of concentration and pH. I have grown a couple crystals that
were so pH sensitive that pH changes of less than 0.1 had profound
effects. Of course, this is not the best position to be once you are
trying to grow multiple crystals from different preps for data
collection. Nor would you want all of that phosphate in your crystal
during heavy atom screening. But, at least in one case, I was able to
grow the same crystal in a new buffer system and do MIR. I would
probably have not stumbled upon that final condition without going
through the phosphate route. So, in my experience, a phosphate screen
is always worth attempting, especially with highly charged samples.

Best wishes,

Tom Huxford.
San Diego State University

LEGAL DISCLOSURE: The author of this post does not own shares in
PHOSPHATE nor will he benefit financially from your use of PHOSPHATE as
a crystallization screening reagent. Past performance does not insure
future success. Blah blah blah...

On Jul 1, 2008, at 9:32 PM, Karakas, Erkan wrote:

> Hi,
>
> I am working on an acidic protein with a pI value around 4.5.
> Although I can obtain proteins with decent solubility, I haven't been
> able obtain crystals. I was wondering if anyone would have some
> suggestions to increase the chances for crystallization.
>
> Thanks,
> Erkan
>
>

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

Previous message:
Subject: FW: .getTwoTheta() in DiffractionImage library
From: "Remacle, F (Francois)" f {- dot -} remacle {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-07-02

Next message:
Subject: Job
From: Sergiy Klyuyev klyuyev {- at -} UAB {- dot -} EDU
Date: 2008-07-03