I say (of course I would!) why not try co-crystallization with random microseeding using the crystals with the original ligand?
It usually allows you to control the number of crystals per drop too On 18 October 2012 15:59, Dmitry Rodionov <d.rodio...@gmail.com> 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 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
