The literature conditions for SARS-CoV-2 mpro are quite robust, particularly the condition that formed the foundation of the Diamond XCHEM fragment screening campaign <https://www.nature.com/articles/s41467-020-18709-w#Sec8>. The base condition was used for 6YB7 <https://www.rcsb.org/structure/6YB7>, as the condition had DMSO in from the start it proved very amenable to soaking and cocrystallisation.
To improve crystal morphology multiple rounds of seeding were required with fine tuning of precipitant concentrations. Seed stocks proved variable so it was necessary to flash freeze seed stocks and then work from the frozen stock to get consistent results. Starting protein quality is obviously very important. On Mon, 30 Jun 2025 at 20:31, Edward Snell < 000114dd62d93b35-dmarc-requ...@jiscmail.ac.uk> 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 <esn...@hwi.buffalo.edu> > > The University at Buffalo Hauptman-Woodward Institute > 700 Ellicott Street | Buffalo, NY 14203-1102 > > Website: https://snelllab.website/ > > > > [image: hwi-logo-primary-horizontal] > > > > > > > > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> *On Behalf Of *Mohit > Bhardwaj > *Sent:* Monday, June 30, 2025 3:18 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *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 > -- ---------------------------------------------------------------------------------------------------------------------------- (Christopher) David Owen cdavido...@gmail.com No trees were killed to send this message, but a large number of electrons were terribly inconvenienced. ######################################################################## 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/
