All great suggestions and a few new ones I haven't used before (thanks Emmanuel and Frank).
I agree with Emmanuel, a fine screen should definitely be first port of call, change the precipitant to go higher and lower than the initial hit (I normally vary the PEG in 2% jumps, in a 24 or 48 well screen), and also the pH by 0.5-1 points above and below. I would also try different temperatures (higher & lower), and different protein:well ratios (1:1, 1:2, 2:1). Seeding is also my favourite method for optimisation, I usually seed into my normal fine screen (with a 1:2:2, seed:protein:well ratio, if working from an initial 1:1 ratio), and sometimes a new screen with greatly reduced precipitant, but the former has always worked better in my microcrystal cases. There are a lot of suggestions for reducing the protein concentration, I agree, but just to note I once had a case where just diluting the protein with buffer didn't help as the nucleation points didn't break down (tried to reduce 8mg/ml to 4 and 2mg/ml). Instead we did a fresh prep and made sure the concentration didn't go above 4mg/ml throughout the whole prep, and this worked for us (with a 2:1 protein:well ratio). Are there any cysteines in the protein? Sometimes adding more reducing agents helps reduce nucleation. Also sometimes adding a ligand can help improve protein crystal size and quality. Good luck, let us know if anything works! Nicola ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Emmanuel Saridakis <e.sarida...@inn.demokritos.gr> Sent: 12 July 2017 13:55:31 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallization optimization Dear All, Fine-tuning protein and precipitant concentration is of course the first line of approach, followed by both rMMS and streak-seeding. I would like to remind you of a far less popular but often successful in my hands, optimisation technique: It consists in incubating the trial at the condition that gives the ugly crystals for some time (shorter than the time it takes for the ugly crystals to appear), and then changing the condition to one of lower supersaturation for the growth to proceed. That can be done either by diluting the reservoir with water (vapour diffusion) or diluting the drop with buffer (microbatch), or by just transferring your plate to a different (usually higher) temperature. Described in more (but perhaps unecessary for most practical purposes) detail in: E. Saridakis, P.D. Shaw Stewart, L.F. Lloyd and D.M. Blow. Acta Crystallogr. (1994) D50, 293. E E. Saridakis and N.E. Chayen. Protein Science (2000) 9, 755. Best, Emmanuel ________________________________ De: "Alun R Coker" <alun.co...@ucl.ac.uk> À: "CCP4BB" <CCP4BB@JISCMAIL.AC.UK> Envoyé: Mercredi 12 Juillet 2017 15:40:10 Objet: Re: [ccp4bb] crystallization optimization Hi Everyone, Franks point is really interesting. We routinely reduce the protein concentration when we see too many precipitated wells, but we never dilute the screen. Has anyone tried this? All the best, Alun On 12/07/17 08:48, Frank von Delft wrote: The point I was failing to make: reducing either protein or precipitant concentration will indeed reduce nucleation, but often won't get you bigger or more single crystals: it will just make the appearance of crystals less reliable. The way to get big single reliable crystals is to increase protein and greatly reduce precipitant. (Even better: do seeding. Like Vicky said. Incredible how often people don't bother to do seeding, yet it solves so many problems.) phx On 12/07/2017 07:50, Vicky Tsirkone wrote: Dear Frank, I may see in the attached pic several nucleation points and a considerable amount of microcrystals. Based to my knowledge decreasing the concentration of the precipitant and/or the protein concentration would be a reasonable approach to refine the initial hits. By checking the diagram as you correctly mentioned you may see that the fine tuning of protein and precipitant concetration may lead to the desirable result without reaching the precipitation zone. Patrick just check your screens. Just a rule of thumb, if you see precipitation in the ~60% of your drops then you should definitely reduce the protein concentration. ps dont forget to try the streak seeding, as well. Have a nice day and again good luck. Vicky On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft <frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>> wrote: Actually, you should try increasing the protein concentration - a lot. But be prepared to drop the precipitant concentration to almost nothing (1 or 2% isn't "low"). To understand why, look at the phase diagram and what we assume about vapour diffusion. (Which I'm assuming is what you're doing.) On 12/07/2017 06:28, Vicky Tsirkone wrote: Dear Patrick, You may reduce the protein concentation, as well. Another option could be the streak seeding by exploiting the drop of your initial condition. Good luck, V.T. On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart <patr...@douglas.co.uk<mailto:patr...@douglas.co.uk>> wrote: Microseed them into two or three random screens. Search for MMS and rMMS online. Good luck Patrick On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com<mailto:519198...@163.com>> wrote: hello everyone! I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my protein grow small needle like crystals, how can i optimize it to get bigger crystals? the attach is the crystals figure. thanks in advance sincerely Liuqing Chen -- patr...@douglas.co.uk<mailto: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<https://emea01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.douglas.co.uk&data=01%7C01%7CNicola.1.evans%40KCL.AC.UK%7C469379992ad14ccf714508d4c92dc263%7C8370cf1416f34c16b83c724071654356%7C0&sdata=N87sAimapgEuK6dTMgj%2Bmasferzl7THM6thwgqqCRhc%3D&reserved=0> Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034<tel:%28877%29%20225-2034> Regd. England 2177994, VAT Reg. GB 480 7371 36 -- Dr Alun R. Coker Senior Lecturer Wolfson Institute for Biomedical Research University College London The Cruciform Building London WC1E 6BT Tel: 020 7679 6703 Ext 46703 Web: www.ucl.ac.uk/pxmed<https://emea01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.ucl.ac.uk%2Fpxmed&data=01%7C01%7CNicola.1.evans%40KCL.AC.UK%7C469379992ad14ccf714508d4c92dc263%7C8370cf1416f34c16b83c724071654356%7C0&sdata=VP6psrrxD65EEhwN2YtJLfbppk0qokbAxou%2BROUjH88%3D&reserved=0>
