Hi Nicholas,
We have got the binding data from 4 different assays - alpha
screen, FP, MST and SPR, though the values are not exactly the
same in all.
I may have to do the studies with crystallization condition also
in order to re-confirm.
Thanks
Regards
Kavya
> Hi Kavya,
> There are some excellent suggestions in this thread. The one thing I
> would
> add is how convinced are you of the binding data? There are many sources
> of artifact when looking at high ligand concentrations in binding assays.
> It may be that your empty crystal structures are "real" results and your
> assay data was garbage.
>
> In any event, fragment crystallography is challenging and I'm always very
> happy with any success because failure rate can be very high.
> Good luck,
> Nick
>
>
>
> On Wed, Jan 6, 2016 at 12:52 PM, Kavyashree Manjunath <
> [log in to unmask]> wrote:
>
>> Dear Enrico,
>>
>> Yes I used ethylene glycol as cryoprotectant.
>> I did add the same concentration of small molecule
>> in the cryo also. I can't reach 1000x because of
>> DMSO limitation. I generally don't go beyond 15% DMSO
>> in the soaking solution or cryo, as it might damage the
>> crystal.
>>
>> Thank you
>> Regards
>> Kavya
>>
>> > 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
>> >
>> http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
>> >
>>
>>
>>
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