Re: [ccp4bb] SUMMARY: losing zinc during crystallization

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

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From: Sheemei slok {- at -} PURDUE {- dot -} EDU
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Subject: Re: SUMMARY: losing zinc during crystallization
From: Sue Roberts suer {- at -} EMAIL {- dot -} ARIZONA {- dot -} EDU
Date: 2008-10-02

Hello

I wish to thank everyone for all the helpful replies. A summary
follows:

While some expressed surprise at the zinc lability, others related
tales of difficulty keeping zinc in a protein.

Suggested ways of overcoming the problem included:
1) Use of TCEP as a reducing agent
2) Allowing Zn to leave and filling the site with Au or Cd
3) Limiting oxygen during crystallization - crystallization in a glove
bag or degassed solutions.

One person noted that TCEP decomposition products include phosphate
and as a result ZnPO4 crystals can form.

I'm off to try suggestion (3). Hope it works and I don't have to
resort to suggestion (2).

Thanks again for the help.

Sue

>>
>> Hello Everyone
>>
>> I've been trying to crystallize a zinc-containing enzyme for what
>> seems to me to be an eternity. The protein contains stoichiometric
>> zinc (1 zinc/ protein monomer) when isolated and the zinc is required
>> for activity. Each crystal we've obtained has lost the zinc and
>> contains a disulfide bond between two cysteine residues that should
>> be
>> zinc ligands (based on structures of similar proteins).
>>
>> We've tried crystalizing in the presence of reducing agents,
>> crystallizing with substrate analogs, and supplementing the
>> crystallization drops with zinc with no success (and combinations of
>> these approaches). We've obtained a variety of crystals and
>> determined structures, but none contain any zinc.
>>
>> Attempts to insert zinc into the crystal (zinc + reducting agent or
>> zinc alone) have not been successful.
>>
>> Does anyone have any tricks to suggest that might help?
>>
>> Thanks in advance.
>>
>> Sue
>>
>> Dr. Sue A. Roberts
>> Biochemistry & Molecular Biophysics
>> University of Arizona
>> 520 621 8171
>> suer@email.arizona.edu
>>
>

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
suer@email.arizona.edu

CCP4bb navigation

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

Previous message:
Subject: post-doctoral or graduate student position
From: Sheemei slok {- at -} PURDUE {- dot -} EDU
Date: 2008-10-02

Next message:
Subject: Re: Reading the old literature / truncate / refinement programs
From: Pavel Afonine pafonine {- at -} LBL {- dot -} GOV
Date: 2008-10-03