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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