Re: [ccp4bb] removal of sulfate ion from the active site

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

Previous message:
Subject: Re: removal of sulfate ion from the active site
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2007-05-25

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Subject: AW: removal of sulfate ion from the active site
From: Herman {- dot -} Schreuder {- at -} SANOFI-AVENTIS {- dot -} COM Herman {- dot -} Schreuder {- at -} SANOFI-AVENTIS {- dot -} COM
Date: 2007-05-25

Subject: Re: removal of sulfate ion from the active site
From: Marius Schmidt marius {- dot -} schmidt {- at -} PH {- dot -} TUM {- dot -} DE
Date: 2007-05-25

Before a buffer exchange I would increase ligand concentration,
if possible.
Just get the binding constant and the concentration of the protein
in the crystal and CALCULATE the ligand concentration which is
needed to achieve 99.9 % saturation. With moderate binding this
will quickly bring you to the 100 th of mmol/l range. If this is
true 10 mmol/l of ligand will never show up in the electron density.

Also: You can also try ammonium phosphate to get rid of sulfate. However,
I think the problem persists because you might have now a phosphate
in the active center.

> Hi all,
>
> I have crystals of the apo enzyme growing from 1.7M ammonium sulfate
> condition. After soaking with 10mM ligand (substrate analog) which has a
> phosphate group, the ligand did not enter the active site of the enzyme
> because of the competition of sulfate ion and phosphate group. So I am
> wondering if anyone knows how to get rid of ammonium sulfate from
> crystal before soaking it with the ligand solution? Thanks a lot.
>
> Simon

PD Dr. habil. Marius Schmidt
Physikdepartment E17
Technische Universitaet Muenchen
James Franck Strasse
85747 Garching/Germany
email: marius.schmidt@ph.tum.de
http://users.physik.tu-muenchen.de/marius/
phone: +49-(0)89-2891-2550
fax: +49-(0)89-2891-2548

CCP4bb navigation

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

Previous message:
Subject: Re: removal of sulfate ion from the active site
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2007-05-25