Hi Marek, I had a similar problem before, which the active site was partially occupied by a PEG molecule and my ligand couldn't get into the site. Sequential soaking-out can solve my problem; basically you need to slowly remove the PEGs from the crystal (in order to balance the osmotic shock): Stepwise transfer your crystal into three different drops of reservoir solution containing increasing concentrations of glycerol but decreasing PEG concentration in the presence of substrate for 1 min each, say your crystal grown in 20 % PEG: e.g. Solution (1) = 15 % PEG, 5 % glycerol + 0.5 mM substrate; (2) = 10 % PEG, 10 % glycerol + 0.5 mM substrate; (3) = 20 % glycerol only + 0.5 mM substrate. And of course you can try different combinations. It works for me nicely and is repeatable.
HTH, Matt 2009/6/9 <herman.schreu...@sanofi-aventis.com> > Dear Marek, > A lot may happen: Your active site might be blocked by PEG or crystal > contacts, or your sugar substrate gets converted into product and leaves the > active site. In your case I would try the following: > > - soak or cocrystallize with a non-hydrolizable suger analog > - soak with as high a substrate concentration as you can get (preferably > saturated) > - try crystallizing with larger pegs (20k) which may not enter the active > site. > > Good luck! > Herman > > ------------------------------ > *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of > *Marek > Frischerkase > *Sent:* Tuesday, June 09, 2009 1:56 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] soaking problems > > Dear all, > > I tried to soak substrate (sugar) into my enzyme-crystals. After solving > the structure I found PEG molecules instead of substrate in the active > site. Does this mean that the active site is blocked and the substrate isn´t > able to get into the enzyme-crsytals anymore? I used PEG 1000 in my > crystallisation setups. Cocrystallisation showed no success so far. > > Can anyone give some suggestion what I should do? > > Thanks a lot, > > Marek > > -- ---------------------------------------------------------------------------- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester ----------------------------------------------------------------------------
