Hi Mohit, I was able to get crystals of C111S-PLpro using microbatch under oil method in crystal screen of hampton research. Protein concentration was around 10 mg/ml. Crystals diffracted to 2.5 A at NSLS-II.
*Dr. Amit Gaur, * *Department of Biomedical Science,* *College of Medicine,* *Florida State University,* *1115 W Call St, Tallahassee, FL,32304* On Mon, Jun 30, 2025 at 3:30 PM Edward Snell < [email protected]> wrote: > Hi Mohit, > > > > I would agree with the temperature and seeding comments by Emmanuel. > Although not proven for diffraction characteristics, volume ratio and > temperature can be effective optimizing strategies – see Luft et al, 2007 ( > https://hwi.buffalo.edu/wp-content/uploads/2016/12/dvrt.pdf). > > > > I noticed in looking at a number of models in the PDB that dimethyl > sulfoxide (DMSO) seems to be significant in the high-resolution models and > does seem to form key contacts in packing diagrams. Although it’s probably > there to solubilize some of the ligands tested, it does seem to be ordered > in the electron density, bridges chains and probably stabilizes them, and > could be a useful component to add to your current conditions. > > > > It’s not clear from your comments if you had more than one initial > crystallization condition that you optimized, but I’d also recommend > optimizing ‘crappy’ crystals that appear in initial screens if they are > from chemically distinct conditions. A little optimization can go a long > way in going from visually poor to diffraction good crystals. > > > > I think you are heading in the right direction, and the crystals you do > have are beautiful. I presume you have tried a microfocus beamline, but if > not, that is also a good approach. > > > > Best of luck, > > > > Eddie > > > > *Edward Snell Ph.D.* > > Professor, Materials Design and Innovation | University at Buffalo, SUNY > > Director | NSF BioXFEL Science and Technology Center > > Fellow of the American Crystallographic Society – The Structural Science > Society > > p: +1 716 898 8631 | f: +1 716 898 8660 > e: esnell@ buffalo.edu <[email protected]> > > The University at Buffalo Hauptman-Woodward Institute > 700 Ellicott Street > <https://www.google.com/maps/search/700+Ellicott+Street+%7C+Buffalo,%0D%0A+NY+14203-1102?entry=gmail&source=g> > | > <https://www.google.com/maps/search/700+Ellicott+Street+%7C+Buffalo,%0D%0A+NY+14203-1102?entry=gmail&source=g> > Buffalo, NY 14203-1102 > <https://www.google.com/maps/search/700+Ellicott+Street+%7C+Buffalo,%0D%0A+NY+14203-1102?entry=gmail&source=g> > > Website: https://snelllab.website/ > > > > [image: hwi-logo-primary-horizontal] > > > > > > > > *From:* CCP4 bulletin board <[email protected]> *On Behalf Of *Mohit > Bhardwaj > *Sent:* Monday, June 30, 2025 3:18 AM > *To:* [email protected] > *Subject:* [ccp4bb] Crystallization of SARS-CoV-2 3CLpro and Mutants – > Seeking Advice on Crystal Optimization > > > > Dear members, I am currently working on the crystallization of the main > protease (3CLpro) of SARS-CoV-2, focusing on both the wild-type and several > point mutants. Initial crystallization trials were c > > Warning! This message was sent from outside your organization and we were > unable to verify the sender. > > sophospsmartbannerend > > Dear members, > > I am currently working on the crystallization of the main protease > (3CLpro) of SARS-CoV-2, focusing on both the wild-type and several point > mutants. > > Initial crystallization trials were conducted using commercial screens, > including PACT, JCSG+, and INDEX. These trials yielded crystals of various > morphologies—predominantly needles, thin sheets, and flower-like clusters. > To improve crystal quality, I subsequently performed optimization using the > hanging-drop vapor diffusion method by systematically varying PEG > concentration, salt types/concentrations, and pH conditions. Despite these > efforts, the crystals have not shown significant improvement in morphology > or diffraction quality. > > I have also attempted microseeding and macroseeding approaches during > optimization, but the results remain largely the same. I would greatly > appreciate any suggestions or experiences from the community regarding > strategies that could help improve crystal quality in such systems, > particularly with challenging morphologies like needle or sheet-like forms. > > Thank you in advance for your insights. > > Mohit Bhardwaj > > PhD Scholar > > Kusuma School of Biological Sciences > > IIT Delhi, Hauz Khas > > New Delhi- 110016 > > Mobile No. : +91-8895172936, 8700227218 > > > ------------------------------ > > 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/
