Dear Mohit, It seems to me that the first steps of your investigation were performed as they should and in the right order. It is however not infrequent for the usual conditions' fine-tuning and standard macro/microseeding to fail to improve on the original hits. So the question is, where do you take it from there? Two further well-known optimisation strategies are: (1) using temperature as an additional optimisation parameter, and (2) random microseed matrix screening, about which you should search for Patrick Shaw Stewart and Douglas Instruments. If these fail or at any rate do not take you where you'd like, I can recommend two other strategies which are detailed in the following papers: https://doi.org/10.1016/S0006-3495(03)74936-4 (using phase diagrams to uncouple the nucleation and growth stages of crystallisation by diluting the reservoir during incubation - it can also work by shifting temperature during incubation, or by diluting the crystallisation mixture itself if working in batch/microbatch mode); https://doi.org/10.1073/pnas.0504860102 (using porous nucleants, especially Bioglass, marketed under the name "Naomi's Nucleants", in order to nucleate crystals at lower supersaturations, where these crystals may grow with fewer defects) Please keep us posted if any of these worked, more information should go around about such things! Good luck, Emmanuel Principal Researcher, Institute of Nanoscience & Nanotechnology National Centre for Scientific Research "DEMOKRITOS" Ag. Paraskevi, Athens 15341, Greece
-----Original Message----- From: Mohit <[email protected]> To: CCP4BB <[email protected]> Date: Monday, 30 June 2025 11:53 AM EEST 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 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 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/
