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
