MMS often works well for us too! Best wishes, Olga
> On 5 Jun 2018, at 19:24, Artem Evdokimov <[email protected]> wrote: > > 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] > <mailto:[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] > <mailto:[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] > <mailto:[email protected]>> on behalf of Liuqing Chen <[email protected] > <mailto:[email protected]>> > Sent: 04 June 2018 20:57 > To: [email protected] <mailto:[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 > <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 > <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> > > > > -- > [email protected] <mailto:[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 <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 > <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 > <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
