Kavya,
I assume that you use cryoprotection. Correct me if I am wrong.
During this step did you add ligand to your cryosolution?
If not you could wash out the ligand from the crystal.
My advise is to add 1000X ligand also in this step. If it works in the
biochemical experiment
it is likely to work also in your crystal soaking experiment.
To improve your methodology you may read:
149 Ciccone L., Vera L., Tepshi L., Rosalia L., Rossello A. & Stura E.A.
(2015)
Multicomponent mixtures for cryoprotection and ligand solubilization.
Biotechnology Reports 7:120–127.
http://dx.doi.org/10.1016/j.btre.2015.05.008
If it still does not work you may try to obtain alternative crystal forms,
as lattice forces may
priviledge the unbound form.
Enrico.
On Wed, 06 Jan 2016 10:29:20 +0100, Kavyashree Manjunath
<[log in to unmask]> wrote:
> Dear CCP4 users,
>
> I am trying to capture small molecule bound to the protein.
> These small molecules are solubilised in 100% DMSO. And the
> biochemical and biophysical assays shows micromolar affinity
> (two or three digit) towards the protein.
>
> We have extensively tried soaking (max 15% DMSO), long period
> soaking, growing protein crystals in the well precoated with
> the ligand and cocrystallization (max 5% DMSO) experiments,
> but have not succeeded to get any density for the ligand.
>
> The molar ratio that was used in the biochemical experiment
> is ~ 1:1000 (protein:ligand) in order to observe any binding.
> [one of the experiment used was microscale thermophoresis]. In
> these experiments the protein is in nM and small molecule is in
> microM quantities.
>
> For crystallization we are able to reach a maximum of 1:90 (in
> cocrystallization). It is not possible to approach a higher molar
> ratio because of the limitations of DMSO.
>
> So is there a possibility that we can capture the ligand in the crystal?
> I have come across ligands that have been captured even though
> they had mM binding affinities. So can anyone please suggest
> possible ways I can try to capture the small molecule.
>
> Thank you
> Regards
> Kavya
>
>
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
e-mail: [log in to unmask] Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
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