Re: [ccp4bb] protein degraded when setting xtal?

Protein crystallography Programs for crystallography X-ray detectors
Basic tutorials:
Protein Peptide

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 03 March 2008

Previous message:
Subject: radiation damage question
From: Patrick Loll pat {- dot -} loll {- at -} DREXEL {- dot -} EDU
Date: 2008-03-03

Next message:
Subject: Re: protein degraded when setting xtal?
From: Kevin Jude kjude {- at -} BERKELEY {- dot -} EDU
Date: 2008-03-03

Subject: Re: protein degraded when setting xtal?
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2008-03-03

Ngo Duc Tri wrote:
> Dear CCP4 experts,
> I'm working on a kind of cystein peptidase. It shows a little
> degradation after 2 days storing at 4 degree even I tried many kinds
> of protease inhibitors, or glycerol and DTT.
> So I decided to work fast (1 day) then set xtal. After one week I pick
> up some condition and check degradation by SDS-PAGE. It shows heavy
> degradation then I think it's impossible to obtain the crystal if I
> don't find out how to stop the degradation first.
>
> Do you have any idea about my situation? Is it true that I need to
> stop the degradation and how to deal with it?
> Thank you for your advices,
>
> My best regards,
> TriNgo
>
>
If I understand the problem properly, you are attempting to crystallize
a peptidase that autolyzes in solution. If it is not possible to
completely inhibit the protein with an analog or small molecule under
crystallization conditions (and be able to crystallize the complex),
then the most obvious solution to the problem is to prepare an inactive
variant, e.g., with the mutation of the catalytically essential Cys to
Ser. With any luck, the variant protein should still provide valuable
structural and mechanistic context while avoiding pesky autolysis.

Cheers,

--
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowlett@mail.colgate.edu

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 03 March 2008

Previous message:
Subject: radiation damage question
From: Patrick Loll pat {- dot -} loll {- at -} DREXEL {- dot -} EDU
Date: 2008-03-03

Next message:
Subject: Re: protein degraded when setting xtal?
From: Kevin Jude kjude {- at -} BERKELEY {- dot -} EDU
Date: 2008-03-03