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hey Liu ... you got all wonderful suggestion, and they may be time consuming. Meantime, you may work on the crystals in hand, and follow the suggestions as mentioned above. From my experience, i can tell you that crystal age is also an important parameter. I will shoot it as soon as i see it in its (semi)-final size. Leaving crystals in PEG-based solution may not be advantageous. Again, that was my experience. Also, I noticed PEG skin at the top of the drop, and several of the crystals were actually got wrapped in the skin while fishing. Such crystals gave the poorest diffraction. So thats one thing you may look at it.. (in addition to others, as mentioned above) Best wishes -Z Zaigham Khan, PhD Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York On Tue, Jun 5, 2018 at 9:39 PM, khevener <[log in to unmask]> wrote: > > Good suggestions here. In situ proteolysis is a new one on me... > > > Sent from my Verizon, Samsung Galaxy smartphone > > -------- Original message -------- > From: Artem Evdokimov <[log in to unmask]> > Date: 6/5/18 1:24 PM (GMT-06:00) > To: [log in to unmask] > 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 < > [log in to unmask]> 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 <[log in to unmask]> 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. >>> <https://maps.google.com/?q=343+Royal+Parade+%0AParkville.+VIC.+3052+%0AAustralia&entry=gmail&source=g> >>> VIC. 3052 >>> <https://maps.google.com/?q=343+Royal+Parade+%0AParkville.+VIC.+3052+%0AAustralia&entry=gmail&source=g> >>> Australia >>> <https://maps.google.com/?q=343+Royal+Parade+%0AParkville.+VIC.+3052+%0AAustralia&entry=gmail&source=g> >>> Tel +613 9662 7326 >>> Email [log in to unmask] >>> >>> ________________________________________ >>> From: CCP4 bulletin board <[log in to unmask]> on behalf of Liuqing >>> Chen <[log in to unmask]> >>> Sent: 04 June 2018 20:57 >>> To: [log in to unmask] >>> 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 >>> >> >> >> >> -- >> [log in to unmask] 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 >> > > > ------------------------------ > > 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 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1