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). F*70* <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> 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> > 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 > -- 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 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/
