Hi Liuqing Chen You have a lot of good suggestions, but everyone except for Janet has missed out the most important suggestion, and Janet has called it something funny - cross seeding often refers to something else.
You may well have a nucleation problem - that's to say many of your screening experiments may be in the metastable zone of your protein's phase diagram. Try making a seedstock with your existing crystals and adding it to *random *screens. This can be combined with Janet's suggestions 2 (second part), 3, 4, 5, 7, 6 again, and 8. For more information google MMS crystallization, or rMMS crystallization. I hope it works - it very often does! Good luck, Patrick On 4 June 2018 at 21:41, Janet Newman <[email protected]> wrote: > Liuqing Chen, > > Everything that has been said seems reasonable, but there are always > infinite possibilities in crystallisation, so it is more a question of > priorities. Do the easy (or quick) things first. If you have buckets of > prepared protein then what you will try first might be different than if > you have to go and make your protein from scratch each time you set up > crystal trays. > > 1. If you have crystals from an additive screen or seeding - try putting > them in the beam. If you have access to in-plate screening, you can test > the crystals without disturbing them, which will give the best idea of > their native diffraction. Perhaps one of the ugly crystals diffracts well > enough? > > 2. Try cross seeding - seed one or more initial screen(s) (rather than an > optimisation). Try initial screening with seeding at different > temperatures. If you are currently using vapour diffusion, try microbatch. > Or vice versa. > > 3. Try in-situ proteolysis. Add a very small amount of protease to your > protein sample (chymotrypsin is traditional) - say 1:10,000 or 1:1000 > compared to your protein concentration then set up that mixture in initial > screens. > > 4. Is there a ligand/inhibitor/other small molecule that binds? Add that > to your protein before crystallisation. Maybe even try this first! > > 5. Lysine methylation/cysteine modification/other side chain modifications. > > 6. Try using DSF or some other technique to look at your protein's > stability in the formulation it is in. Maybe you can make happier protein > by changing the pH, buffer or salt. > > 7. If you want to be a little more rigorous, take your protein, and a > number of different proteases, and do a time-course experiment with each > protease (add 1:1000 protease to your protein, then take samples at > timepoints - say 0.5 hours, 1 hour, 5 hours and overnight) then run out on > a gel (or analyse by MS) and see if you come down to a stable fragment. If > you do - then use that protease, and while you are waiting for the > crystallisation trials to do their thing, find out what the end points of > the proteolysis fragment are, and make that construct. > > 6. Try a different expression system (different tag, different position of > the tag, cleave/don't cleave the tag). If the protein is produced in a > eukaryotic system (and is glycosylated) try a different one to get > different glycosylation pattern. Try kifunensine treated cells if you are > in a mammalian expression system. > > 8. Try the same protein from other species > > Janet Newman > Principal Scientist / Director, Collaborative Crystallisation Centre (C3) > CSIRO Material Science and Engineering > 343 Royal Parade > Parkville. VIC. 3052 > Australia > Tel +613 9662 7326 > Email [email protected] > > ________________________________________ > From: CCP4 bulletin board <[email protected]> on behalf of Liuqing > Chen <[email protected]> > Sent: 04 June 2018 20:57 > To: [email protected] > Subject: [ccp4bb] suggestion of crystallization optimization > > Hello everyone! > > I get a crystal several months ago, but the crystals diffraction very low > resolution (around 8A) or no diffraction. > My sample buffer is 20mm Hepes ph7.0, 50mm NaCl, my protein pI is 5. > the codition grow crytal is : 30% PEG400, 100mm hepes 7.5, 200mm MgCl2. > > I also tried additive screen, all the crystals appear the same > apparence, even i seeding optimization, have no improve. > the attach is my crystals. > > what should i do next? > > thanks in advance. > sincerely > Liuqing chen > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- [email protected] Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart 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/webadmin?SUBED1=CCP4BB&A=1
