I want to second the recommendation to try microseed matrix screening. I recently had a case of a protein that did not yield any crystals after trying more than 500 conditions. Of those 500, one single condition gave to me what appeared to be crystalline material, but not distinct single crystals. I harvested that well, crushed up the material as best I could to make a seed stock, and then used the seed stock with the first 48 conditions of I think the JCSG+ screen. Came back the next day, checked the trays under the microscope, and was astonished to find at least 10 wells that had gorgeous crystals in them. I harvested a few crystals from different wells, shot them at the Advanced Photon Source, and nearly fainted when diffraction to almost 1 Å popped up on the monitor for almost all of them. So, you could say I’m a believer in random microseed matrix screening now …
Good luck. Matthew --- Matthew J. Whitley, Ph.D. Research Instructor Department of Pharmacology & Chemical Biology University of Pittsburgh School of Medicine ------------------------------ Date: Thu, 17 Dec 2020 21:54:15 +0000 From: David Briggs <[email protected]> Subject: Re: Micro/Macro crystal seeding experience Hi Rafael, there are many potential answers to questions such as this. Here are the first few that spring to mind: 1. Did you test room temperature diffraction? Is it your cryo-protectant that is causing problems. 2. What is your cryo-cooling protocol? Do you just dunk the crystals straight in to crystallisation liquor + 30% glycerol, or do you slowly step up the cryo-protectant concentration? 3. Additive screens are worth a try. 4. Modify the construct (trim termini, tags, or disordered loop regions). 5. Microseed matrix screening to look for alternative conditions. (https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.douglas.co.uk%2Fmms.htm&data=04%7C01%7Cmjw100%40PITT.EDU%7C5f603236dfef49d09b5308d8a2d6b188%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637438390278906705%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=JJ1pHkScwTx8%2FJCZPXY9DYlIzosr9j67alrdsXIseCY%3D&reserved=0) Good luck! Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs ######################################################################## 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/
