Hi Sabine,
Glutaraldehyde crosslinking worked pretty good for various soaks in my
experience.
J. Appl. Cryst. (1999). 32, 106-112 [ doi:10.1107/S002188989801053X ]
A gentle vapor-diffusion technique for cross-linking of protein crystals for
cryocrystallography
C. J. Lusty
Best regards,
Dmitry
On 2012-10-17, at 12:26 PM, Sabine Schneider wrote:
> Hi everyone,
>
> I am trying to get the structure of a protein-ligand complex were I need to
> exchange the ligand which it co-crystallises nicely with.
> Problem: either they crack, disolve, turn brown,... OR they still look very
> nice, well shaped but do not show a single reflection at the synchrotron!!!
>
>
> Here is what I tried so far:
>
> 1) initially stabilising with higher precipitant (here PEG1500) before slowly
> transferring (*) it to the ligand-removal solution (= artifical mother liquor
> with higher PEG, ethylen glycol or glucose, but without initial ligand)
>
> (*) by slow exchange I mean : initially mixing drop solution with
> stabilising/ligand-removal solution and adding it back to the drop stepwise
> before fully transferring it. Or calculation wise I have fully exchange the
> solution to the new solution
>
> 2) here I let them ist over night (if they did not disolve, crack or whatever)
> 3) slow exchange transfer to the artificial ML with the new ligand (10mM),
> left them over night and directly froze them
>
> 'Best' so far (crystals still looking nice but no reflection...) was slow
> exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also
> adding ethylenglycol to the reservoir), let them sit for over night, before
> again slow exchange to the solution with the new ligand in higher PEG and 30%
> ethylen glycol.
>
> As I said here the crystals keep shape, but don't diffract at all anymore.
> Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a
> home source. But already after step one they are sometimes not happy anymore.
>
> Co-crystallisation failed since when I add the ligand, which is not that
> soluble to the purified protein, everything crashed out of solution. I am
> thinking about to test adding the ligand to the diluted protein and
> concentrate it together. But I don't have that much ligand, since the
> synthesis is quite tedious.... The ligand can be dissolved in 30%
> ethylenglycol to ~50mM
>
> Thus I was wondering if someone has done successfully ligand exchange with
> glutaraldehyd stabilised xtals?
> Or any ideas how to stabilise them? I appreciate any ideas or comments!
>
> Sorry for the lengthy email!
>
> Best,
> Sabine
