Janet, Patrick, and others beat me to it :) We have tremendous luck with MMS in cases like this.
I would also suggest looking at 'atypical' additives - the word atypical is really not a good choice since the world of additives is pretty much infinite, but folks too often tend to take the lazy way out and use the few pre-compiled libraries of 'typical additives' that are available commercially. Not to mention that in cases like this a lot of improvement can be achieved by exploring protein concentration as a factor (not the same as drop ratio, that's also a lazy way out but it's not the same) and by switching buffers and salts to other buffers and salts (at the same pH even!). Crystals, protein crystals, with 8A diffraction - you're 65% done. The last 35% can take a year :) Artem - Cosmic Cats approve of this message On Tue, Jun 5, 2018 at 12:58 PM, Patrick Shaw Stewart <[email protected] > wrote: > > 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 >> <https://maps.google.com/?q=343+Royal+Parade+%0D%0AParkville.%C2%A0+VIC.+3052+%0D%0AAustralia&entry=gmail&source=g> >> 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. 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