Thanks a lot for all the excellent suggestions!
Lots more things to try now!

Cheers,
Sabine


On 10/18/2012 12:18 PM, Boaz Shaanan wrote:
> Hi Sabine,
>
> On top of the excellent suggestions of Herman, I was just wondering.   Do you have a structure of the ligand-bound protein? If you do and the ligand bound at crystal contact with another molecule, I would think that it would be hard to get it out without harming the crystals (although not impossible). This may help you decide which way to go for obtaining the ligand-free structure.
>
>   Cheers,
>
>              Boaz
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben-Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> E-mail: [log in to unmask]
> Phone: 972-8-647-2220  Skype: boaz.shaanan
> Fax:   972-8-647-2992 or 972-8-646-1710
>
>
>
>
>
> ________________________________________
> From: CCP4 bulletin board [[log in to unmask]] on behalf of [log in to unmask] [[log in to unmask]]
> Sent: Thursday, October 18, 2012 10:32 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Stabilization of crystals and ligand exchange
>
> Hi Sabine,
>
> The easy experiment to start with, is to take your best conditions (nice looking crystals, no diffraction) and instead of overnight wait 4-8 weeks. Sometimes ligand exchange is slow, or the ligand induces a conformational change which takes a long time to complete in the crystals. There are cases that after a number of weeks, diffraction came back.
>
> However, even if the above experiment works, there will be the nagging uncertainty that the crystal packing may have prevented some completely unexpected, nature or science publication worthy conformational change. There will be no way around at least trying to cocrystallize your ligand. Your chance of success will depend on how your protein and ligand behave:
>
> -your ligand causes your protein to precipitate. Here your chances are slim. You could try to use the ligand as a precipitant by slowly diffusing it in (e.g. in a capillary with some gel to separate the protein and ligand solutions).
> -your ligand is poorly soluble. Here you have better chances. As you mentioned, one can add the dilute ligand to a dilute protein solution and then concentrate the complex. The amount of ligand needed depends on the affinity of the ligand for the protein. To get 90% occupancy, you need a free ligand concentration at least 10 times over the Kd (or ~IC50).
>
> To give an example: if you use for crystallization 10 mg/ml of a 30 kDa protein, your protein concentration is ~0.33 mM. If your ligand has an affinity of 100 nM, you need a free ligand concentration of 1 µM, which is 300 fold less than what you need to saturate all binding sites in the protein. To account for uncertainties in protein concentrations, I would add 0.5 - 1.0 mM Ligand. If you dilute 10 fold, you have 33 µM protein and I would add 50-100 µM ligand, which is still well above the 1 µM free concentration needed. Even with 100 fold dilution, you still just can dilute the ligand with the same factor as the protein and still be well above the required free concentration and you do not need more ligand.
>
> Of course, this only works for high-affinity ligands, for low affinity ligands it is quite a different story. I also would try to use the highest possible ligand concentration, since in many cases, although the ligand should bind in theory, in practise it is quite a different story.
>
> Good luck!
> Herman
>
>
>
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Sabine Schneider
> Sent: Wednesday, October 17, 2012 6:27 PM
> To: [log in to unmask]
> Subject: [ccp4bb] Stabilization of crystals and ligand exchange
>
> 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
>