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

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

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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

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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

Dear Marius and others,
here I would like to comment: The problem with soaking is often not so much the concentration of the ligand, but the amount of ligand needed to fill all binding sites in the crystal. A typical crystal contains about 20 mM protein so one has to add the equivalent amount of ligand. On the other hand, the concentration UNBOUND inhibitor, asuming a 1:1 protein-ligand complex, need only to be in the order of 10-100 times the Kd (90-99% occupancy). Ways to overcome this is to soak in a large volume of mother liquor (containing a large amount of ligand) or to add solid ligand to the mother liquor and let the ligand slowly dissolve and diffuse into the crystal.

Herman

-----Ursprüngliche Nachricht-----
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Marius Schmidt
Gesendet: Freitag, 25. Mai 2007 14:44
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] removal of sulfate ion from the active site

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: Marius Schmidt marius {- dot -} schmidt {- at -} PH {- dot -} TUM {- dot -} DE
Date: 2007-05-25

Next message:
Subject: ccp4i unable to extract data from project Directory
From: Jhon Thomas jhon1 {- dot -} thomas {- at -} GMAIL {- dot -} COM
Date: 2007-05-25