Good suggestions here. In situ proteolysis is a new one on me... Sent from my Verizon, Samsung Galaxy smartphone -------- Original message --------From: Artem Evdokimov <artem.evdoki...@gmail.com> Date: 6/5/18 1:24 PM (GMT-06:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] suggestion of crystallization optimization 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 <patr...@douglas.co.uk> 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 <janet.new...@csiro.au> 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 janet.new...@csiro.au ________________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Liuqing Chen <519198...@163.com> Sent: 04 June 2018 20:57 To: CCP4BB@JISCMAIL.AC.UK 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 -- patr...@douglas.co.uk 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. 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