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: [email protected]
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
________________________________________
From: CCP4 bulletin board [[email protected]] on behalf of
[email protected] [[email protected]]
Sent: Thursday, October 18, 2012 10:32 AM
To: [email protected]
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:[email protected]] On Behalf Of Sabine
Schneider
Sent: Wednesday, October 17, 2012 6:27 PM
To: [email protected]
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
