Re: [ccp4bb] citrate blocks the active site

[Herman . Schreuder]

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

[Pascal Egea]

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