What I’m about to write should be referred as a question rather than an answer. However, it might also help to find the answer to crystallization question discussed here. The good old crystallization diagram so far for me was something that I’d look after successful crystallization story and find in which direction my optimization went. Each condition in every screen is just a point at the diagram. Were on the diagram that point is situated you don’t know because the scales of X and Y axes are unknown. You can find those scales by deliberately setting up similar screens with diluted (or concentrated, or both) of protein sample (Y axis scale) and diluted (mostly) crystallization screen. This is the way I can make use of the crystallization diagram. Unfortunately, often we cannot spare enough protein to do so. In such cases going through different screens and looking for similar conditions sometime allows finding horizontal line on which your crystallization position should be. After this few optimization attempts at different protein concentrations may help finding position on the diagram and clues where to go.
I hope what I just wrote makes sense. If there is a better way of using crystallization diagram I’d love to hear. Regards, Vaheh Oganesyan www.medimmune.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Philippe BENAS Sent: Thursday, July 13, 2017 12:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallization optimization Dear all, I fully agree with all the suggestions, but it seems that no one has raised the issue of the solubility curve changes on the pH. If the dilution of the protein or precipating agent can indeed modify starting and the equilbrium points on the phase diagram, I would also suggest trying various pH as they can change a whole lot of the net protein charge, therefore the corresponding solubility curve and nucleation zone and hence the entire corresponding phase diagram (for more info PubMed search with "Madeleine Riess-Kautt" as keywords, a great scientist who dedidacted her career to understanding of the so-called Hofmeister series). All the best, Philippe ________________________________ Philippe BENAS, Ph.D. Dog in the manger "Un importun survient qui trouble l'intimité, qui arrête l'expansion, qui glace le plaisir, - probablement comme un étranger tombant au milieu d'enfants en train de danser une ronde", Alfred Delvau, Dictionnaire de la langue verte (1866). Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS Faculté de Pharmacie, Université Paris Descartes Case 48 Av, de l'Observatoire F-75270 PARIS cedex 06 +33.1.5373.1599 E-mails: philippe.be...@parisdescartes.fr<mailto:philippe.be...@parisdescartes.fr>, philippe_be...@yahoo.fr<mailto:philippe_be...@yahoo.fr> URLs: http://lcrbw.pharmacie.univ-paris5.fr/<https://clicktime.symantec.com/a/1/SHOanJkxzhaimfWeJjsKhLk-TE_MYWKSUVPPPJ-A7xc=?d=ydHuK2RzVboI0S1f0-VMFO47v0jLw-bGOuPIOXlvgxVovpKjpVbqlqHHe92eD9EXP1yEiYalPcDJ38NT0DPj0JHU1ay-7q9vqP6cG6Jh_Y2NFwbuGut6uA1GqbEf11-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwlOii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgCC5qcLQd_xWchIlNmFOVWl0_LVlkIZxPEbwqt58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D&u=http%3A%2F%2Flcrbw.pharmacie.univ-paris5.fr%2F> , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18<https://clicktime.symantec.com/a/1/SiKnIzlCgLoTAR5pxIlBBAWAsBmNDNdJ41f4ue88mvg=?d=ydHuK2RzVboI0S1f0-VMFO47v0jLw-bGOuPIOXlvgxVovpKjpVbqlqHHe92eD9EXP1yEiYalPcDJ38NT0DPj0JHU1ay-7q9vqP6cG6Jh_Y2NFwbuGut6uA1GqbEf11-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwlOii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgCC5qcLQd_xWchIlNmFOVWl0_LVlkIZxPEbwqt58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D&u=http%3A%2F%2Flcrbw.pharmacie.univ-paris5.fr%2Fspip.php%3Farticle18> ________________________________ ________________________________ De : Patrick Shaw Stewart <patr...@douglas.co.uk<mailto:patr...@douglas.co.uk>> À : CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Envoyé le : Mercredi 12 juillet 2017 17h28 Objet : Re: [ccp4bb] crystallization optimization Alun I agree Frank's point is very interesting - and he intriguingly refers us to the phase diagram. Is the point that Line A is longer than Line B ? Best wishes Patrick [Inline images 2] On 12 July 2017 at 14:40, Alun R Coker <alun.co...@ucl.ac.uk<mailto:alun.co...@ucl.ac.uk>> wrote: 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://clicktime.symantec.com/a/1/25dI3WrmU_REz0R2AqEuuVcDP3s_fHPB3ls19C25QnM=?d=ydHuK2RzVboI0S1f0-VMFO47v0jLw-bGOuPIOXlvgxVovpKjpVbqlqHHe92eD9EXP1yEiYalPcDJ38NT0DPj0JHU1ay-7q9vqP6cG6Jh_Y2NFwbuGut6uA1GqbEf11-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwlOii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgCC5qcLQd_xWchIlNmFOVWl0_LVlkIZxPEbwqt58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D&u=http%3A%2F%2Fwww.douglas.co.uk%2F> Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 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<http://www.ucl.ac.uk/pxmed> -- 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://clicktime.symantec.com/a/1/25dI3WrmU_REz0R2AqEuuVcDP3s_fHPB3ls19C25QnM=?d=ydHuK2RzVboI0S1f0-VMFO47v0jLw-bGOuPIOXlvgxVovpKjpVbqlqHHe92eD9EXP1yEiYalPcDJ38NT0DPj0JHU1ay-7q9vqP6cG6Jh_Y2NFwbuGut6uA1GqbEf11-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwlOii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgCC5qcLQd_xWchIlNmFOVWl0_LVlkIZxPEbwqt58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D&u=http%3A%2F%2Fwww.douglas.co.uk%2F> Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. 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