Vaheh, one thing you can do is set up a microbatch-under-oil plate, varying protein against precipitant, with a nice big variation in both parameters. Then set up exactly the same experiment but with say 20 nl of seed-stock added to each drop. Hey presto that gives you the one dotted and two solid lines that I showed on my diagram, conveniently laid out on two plates. (Convenient also that you happen to have good automation at MedImmune ; )
That kind of thing is very helpful for people who want to grow crystals for xFEL or neutron diffraction - it may not be absolutely essential for routine structure solution. But I always find it helpful to bear in mind, as Frank suggests. Very best wishes, Patrick On 13 July 2017 at 14:49, Oganesyan, Vaheh <oganesy...@medimmune.com> wrote: > 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 <http://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 <+33%201%2053%2073%2015%2099> > > E-mails: philippe.be...@parisdescartes.fr, 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> > *À :* 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 > > > > > > > > > > [image: Inline images 2] > > > > > > > > > > > > > > On 12 July 2017 at 14:40, Alun R Coker <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> 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> 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> 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 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 <01488%20649090> 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 <020%207679%206703> > > Web: www.ucl.ac.uk/pxmed > > > > 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. This > communication is expected to be read and/or used only by the individual(s) > for whom it is intended. If you have received this electronic communication > in error, please reply to the sender advising of the error in transmission > and delete the original message and any accompanying documents from your > system immediately, without copying, reviewing or otherwise using them for > any purpose. Thank you for your cooperation. > -- 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. GB 480 7371 36
