A few ideas, some already mentioned:
1. Try as many different ligands as you can. In the case of substrates,
non-hydrolyzable analogs or transition state analogs might work better.
But try everything you can get your hands on that you'd be happy to get
a structure of.
2. Try soaking the ligand into the crystals
- Vary time, 2hrs to 5days+
- Vary ligand concentration
- Add ligand to cryo-protectant solution
- Try different cryos, DMSO is a good one if it works for the
crystal
3. Co-crystallization with ligand present
- known conditions
- rescreen protein in partial factorials with streak seeding
from existing crystals
- try different solvents for ligand and different solvent
concentrations (up to ~10% final?)
- try different ligand concentrations
4. Add ligand earlier
- during protein concentration
- before last purification step
- at cell lysis
- during fermentation
- might require readding ligand at various steps
Shane Atwell
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
> Behalf Of David Briggs
> Sent: Thursday, September 13, 2007 1:03 AM
> To: [email protected]
> Subject: Re: [ccp4bb] Tough ligand to bind?
>
> Hi Nian,
>
> This isn't a solution but I remember seeing an excellent talk
> by Annie Hassell at the CCP4 study weekend a couple of years back.
>
> The paper from that talk may help you out.
>
>
> Acta Cryst. (2007). D63, 72-79 [
> doi:10.1107/S0907444906047020 ] Crystallization of
> protein-ligand complexes A. M. Hassell, G. An, R. K. Bledsoe,
> J. M. Bynum, H. L. Carter, S.-J.J. Deng, R. T. Gampe, T. E.
> Grisard, K. P. Madauss, R. T. Nolte, W. J. Rocque, L. Wang,
> K. L. Weaver, S. P. Williams, G. B. Wisely, R.
> Xu and L. M. Shewchuk
>
> Synopsis: Methods presented for growing protein-ligand
> complexes fall into the categories of co-expression of the
> protein with the ligands of interest, use of the ligands
> during protein purification, cocrystallization and soaking
> the ligands into existing crystals.
>
>
> HTH,
>
> Dave
>
>
> On 12/09/2007, Joe Krahn <[EMAIL PROTECTED]> wrote:
> > I am working with a protein/ligand complex that is very
> resistant. to
> > getting a stable complex. The first thing to try is to go to
> > saturating ligand concentrations, even if it seems that it is
> > excessive. It is possible that binding affinity is
> significantly different.
> >
> > My biggest problem is precipitant interfering with binding,
> combined
> > with relatively low substrate solubility. I tried transferring to a
> > different condition, but it seems that most precipitating
> agents have
> > a solubility affect on the ligand as well. I ended up screening
> > conditions just to understand ligand solubilities. I even
> tried things
> > like DMSO additives.
> >
> > Eventually, I found conditions where the ligand is fairly
> soluble, but
> > also stabilizes the protein. I found that ethylene glycol helped
> > binding even though glycerol inhibited it. I also changed
> the pH, and
> > did the soak at room temperature instead of in the cold room. Even
> > then, I used saturating ligand concentrations.
> >
> > Joe Krahn
> >
> > Nian Huang wrote:
> > > Dear All,
> > >
> > > I have been trying to get a protein-ligand complex crystal for a
> > > long time. Here, ligand is either substrate or product for this
> > > kinase. I have tried many methods: soaking with apo-crystal,
> > > co-crystal with different concentration of ligand and
> screen for new
> > > conditions. I got a couple of co-crystals with the new
> conditions,
> > > but still no ligand was found in the active site.
> > >
> > > Anybody has some new ideas that I can try? I have been using
> > > glycerol as my cryoprotectant. Could cryoprotectant have some
> > > negative effect on co-crystal? I will try to collect some data at
> > > room temperature in the future.
> > >
> > > Thank you in advance for your suggestion.
> > >
> > > Nian
> > >
> > > Dept of Biochemistry
> > > UT Southwestern Medical Center
> >
>
>
>
> --
> ---------------------------------------
> David Briggs, PhD.
> Father & Crystallographer
> www.dbriggs.talktalk.net
> iChat AIM ID: DBassophile
> ---------------------------------------
> Anyone who is capable of getting themselves made President
> should on no account be allowed to do the job. - Douglas Adams
>
