Dear Amit,
If you will decide to try soakings, you could also try to dehydrate the
drop to increase ligand concentration, as well as avoiding to wash the
crystals in cryo-protection solutions, as described here
(https://journals.iucr.org/j/issues/2022/02/00/ap5042/index.html).
Good luck!
D
On 03/10/2024 14:42, Eta A Isiorho wrote:
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Hi Dr. Gaur,
A few questions:
1. Do you know the kinetics (binding constants, etc) of the drug and
the protein?
2. Do you know how soluble the drug is in DMSO (is 10 mM the most
concentrated?)
3. Is the drug soluble in a mixture of DMSO and water (70% DMSO)?
4. Have you tried crystal soaking experiments?
DMSO can poison crystal formation and it can also enhance it as an
additive as well as a cryoprotectant. You’ll have to determine how
much DMSO you can add and still obtain diffractable crystals under 2.5 Å.
My suggestions would be:
* Take an apo crystal and transfer it to a drop with the amount of
DMSO you plan on adding in your mother liquor and observe (does it
wiggle, did it dissolve, does it still diffract?)
* Grow your apo crystal in the presence of DMSO and shoot it (did
the crystal grow, diffract, etc)
* Get the most concentrated sample of your compound and do
o Co-crystallization experiments
o Soaking experiments
* If DMSO is donking up your experiment, try a different solvent, or
a lower concentration of DMSO in water.
Best,
eta
***
Eta A. Isiorho, Ph.D.
Research Assistant Professor
Macromolecular Crystallization Facility Manager
CUNY Advanced Science Research Center
85 Saint Nicholas Terrace, 3.352B/3.134
New York, NY 10031
eisio...@gc.cuny.edu
*From: *CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of amit
gaur <cdriamitg...@gmail.com>
*Date: *Wednesday, October 2, 2024 at 2:51 PM
*To: *CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
*Subject: *[ccp4bb] Co-Crystallization with drug molecule
Hi everyone,
I am trying to crystallize a protein with a drug molecule. The protein
concentration is 15.5 mg/ml, the drug stock concentration is 10 mM,
and the drug is dissolved in DMSO. I am adding the drug to a final
concentration of 1 mM in 100 ul of protein, and the DMSO volume is 10
ul for Co-crystallization. I want to know how much DMSO is permissible
during co-crystallization with the drug and if DMSO can poison crystal
formation. I have not been successful in getting crystals with
inhibitors till now, but I obtained crystals of protein without DMSO,
and those diffracted to 2.5A.
Thanks,
*Dr. Amit Gaur,*
*Research Scientist*
*Center for Biotechnology and **Interdisciplinary Studies,*
*Rensselaer Polytechnic Institute,*
*1623 15th Street, Troy, NY, 12180*
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