Hi Sabine,
I also have good experience with crosslinking crystals with
glutaraldehyde to stabilise them for soaks with ligands that were only
soluble in DMSO (http://dx.doi.org/10.1016/j.jmb.2006.06.007). More
often than not it destroys the crystals, I think, but it's worth a try.
If you can get crystals of the ligand-free protein which you then
crosslink, then you might be able to add the ligand in ethylenglycol
without destroying the crystals. We added the glutaraldehyde straight to
the crystals, and that worked just fine, but the vapor-diffusion
technique does sound like a much gentler method.
Exchanging one ligand for another is probably a different matter
altogether. (Is the affinity of your second ligand higher? What's the
off-rate of the first one?)
Good luck!
Julia
On 10/18/12 4:59 PM, Dmitry Rodionov wrote:
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
--
Dr. Julia Griese
Stockholm Center for Biomembrane Research
Department of Biochemistry and Biophysics
Stockholm University
106 91 Stockholm
Sweden
phone: +46-(0)8-162 746
email: [email protected]
