Wholeheartedly agree on all these points! Works wonders. -- Kelvin Lau https://people.epfl.ch/kelvin.lau
Protein production and structure core facility - PTPSP EPFL SV PTECH PTPSP AI 2146 (Bâtiment AI) Station 19 CH-1015 Lausanne Switzerland Email: kelvin....@epfl.ch<mailto:kelvin....@epfl.ch> Phone: +41 21 69 30267<tel:+41%2021%2069%C2%A030267> If unreachable: +41 21 69 34494<tel:+41%2021%2069%C2%A030267> On 18.12.20, 11:48, "CCP4 bulletin board on behalf of Patrick Shaw Stewart" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of patr...@douglas.co.uk<mailto:patr...@douglas.co.uk>> wrote: Hi Rafael Just to amplify Matthew and David's point about random microseed matrix screening - 1. The idea is you add seeds to RANDOM SCREENS - not everyone understands this! People find it very hard to "let go" of the first conditions they see that give them crystals. 2. I now have the opposite point of view: if I have a choice between seeding and non-seeding conditions I always try to work with the seeding ones. The reason is, you have far more control, because you can dilute the seed stock and "dial up" the number of crystals that you want per drop. 3. Try cross-seeding with crystals of homologous proteins, mutants etc, even crystals with 20% sequence identity can work. 4. Seeds are not all alike - sometimes seeds with a particular unit cell work but other seeds (different unit cell) don't. See ref** 5. Just throw seeds in with a new project - at worst you're running another screening experiment. ** Acta Cryst.<https://journals.iucr.org/f> (2014). F70<https://journals.iucr.org/f/contents/backissues.html>, 1107-1115, Obmolova et al. Crystals of Fab 3-53/L6, grown by self-seeding, diffracted to 2.8Å (Figure 4b). However crystals of the same protein grown by cross-seeding (with crystals of a homologous Fab) looked completely different and diffracted to 2.3Å (figure 4c). https://scripts.iucr.org/cgi-bin/paper?nj5193 (open access). Your project shouts "seed me". Hope it works Patrick On Thu, Dec 17, 2020 at 10:27 PM Whitley, Matthew J <mjw...@pitt.edu<mailto:mjw...@pitt.edu>> wrote: 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 <david.bri...@crick.ac.uk<mailto:david.bri...@crick.ac.uk>> 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<http://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 -- patr...@douglas.co.uk<mailto:patr...@douglas.co.uk> Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ________________________________ 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/
