Re: [ccp4bb] citrate blocks the active site
Dear Skkannu, since citrate is (a) necessary for crystal growth and stability and (b) blocks the active site I am afraid you will have to find a completely different crystal form. This means start screening all over again for crystallization conditions of your enzyme in the presence of a sufficiently large amount of ligand. And make sure your ligand is not prcessed by the enzyme. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Samy kannu Sent: Thursday, June 11, 2009 4:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] citrate blocks the active site Dear all, One of my enzymes crystallized in 0.1 M citric acid pH 3.5 and 25% PEK 3350. The structure solution reveals, citrate molecule is occupying the active site. Moreover citrate mediates the dimer formation. I tried crystallization in the same condition with ligand using soaking with different concentrations of ligands and lower concentration of citric acid (below 0.5 M of citric acid, crystals are broken). Cocrystallization also tried. I collected several data sets with these combinations. The Structure solutions from all these data sets give only citrate complex. I found crystals only in citrate condition. Any suggestions !! Thanks. Skkannu
Re: [ccp4bb] citrate blocks the active site
Dear Samy, You mentioned that your enzyme crystallizes in 0.1M citric acid pH3.5 plus 25% PEG 3,350. I wonder if you have tried to systematically scan for other carboxylic acids as buffer/co-precipitating salts. I would suggest you to try the serie of carboxylic acids, citrate, acetate, formate and also the magic carboxylate malonate; a little bit like the Hofmeister series. You maybe able to find a surrogate to citrate that will able you to either soak your crystals or co-crystallize successfully with your substrate(s). Hope this helps Cheers, Pascal Egea University of California San Francisco Department of Biochemistry and Biophysics
[ccp4bb] citrate blocks the active site
> > Dear all, > > One of my enzymes crystallized in 0.1 M citric acid pH 3.5 and 25% PEK > 3350. The structure solution reveals, citrate molecule is occupying the > active site. > Moreover citrate mediates the dimer formation. > I tried crystallization in the same condition with ligand using soaking > with different concentrations of ligands and lower concentration of citric > acid (below 0.5 M of citric acid, crystals are broken). Cocrystallization > also tried. > I collected several data sets with these combinations. > The Structure solutions from all these data sets give only citrate complex. > > I found crystals only in citrate condition. > > Any suggestions !! > > Thanks. > Skkannu > > > > > >