Hi Amit, the questions and suggestions by Eta are important ones and I would like to extend them a little bit.
For your apo crystals, instead of adding DMSO in one go, transfer the crystal to a drop with 5% DMSO and wait, if it survives, transfer to 10%, to 20% and finally to 30%. Then, do not only test the apo form in the presence of xx % DMSO (whatever the crystals tolerated), determine the structure model and 1) try to locate DMSO molecules in the ED map (are these in the binding site?) 2) is the expected drug binding site accessible for the drug or do crystal contacts prevent the binding? As for as the drug itself is concerned, a concentration of 19-fold the Kd would theoretically give a 95% occupancy. Good luck, J. __ Dr. math. et dis. nat. Jeroen R. Mesters University of Lübeck https://orcid.org/0000-0001-8532-6699 Am 03.10.2024 um 15:42 schrieb Eta A Isiorho <etisi...@gmail.com>: 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 * Co-crystallization experiments * 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<mailto:eisio...@gc.cuny.edu> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of amit gaur <cdriamitg...@gmail.com<mailto:cdriamitg...@gmail.com>> Date: Wednesday, October 2, 2024 at 2:51 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <CCP4BB@JISCMAIL.AC.UK<mailto: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 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
