Re: [ccp4bb] crystallization optimization

t58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D=http%3A%2F%2Flcrbw.pharmacie.univ-paris5.fr%2Fspip.php%3Farticle18> > -- > > > > > -- > > *De :* Patrick Shaw Stewa

Re: [ccp4bb] crystallization optimization

Of Oganesyan, Vaheh Sent: Thursday, July 13, 2017 9:50 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallization optimization 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

Re: [ccp4bb] crystallization optimization

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.

Re: [ccp4bb] crystallization optimization

and Wales: Registration number 1794026 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debanu Das Sent: 12 July 2017 22:38 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallization optimization Yes, we have done this (addition of water to dilute screen reagents

Re: [ccp4bb] crystallization optimization

Yes, we have our screenmaking robot programmed to set 50% diluted screens and frequently employ this when a large fraction of the undiluted screens result in precipitation. We have found quite a few productive hits this way, as some previously grungy wells often present crystals when diluted.

Re: [ccp4bb] crystallization optimization

Yes, we have done this (addition of water to dilute screen reagents in the well) and also try it now in some cases and in fact, this is also the rationale behind Hampton's Crystal Screen Lite. Best, Debanu -- Debanu Das > On Jul 12, 2017, at 9:01 AM, Alun R Coker wrote: >

Re: [ccp4bb] crystallization optimization

ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>> Date: Wednesday, July 12, 2017 at 10:54 AM To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] crystallization optimization Yes, exactly.

Re: [ccp4bb] crystallization optimization

So, if we have a commercial 96 well screen where more than around 60% of the drops precipitate. It may be worth diluting the whole screen say (30ul screen and 20ul water in each well) and repeating . rather than diluting the protein. Has anyone ever tried this? All the best, Alun On

Re: [ccp4bb] crystallization optimization

Yes, exactly. Thanks for doing the Right Thing and posting the actual diagram. On 12/07/2017 16:26, Patrick Shaw Stewart wrote: 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

Re: [ccp4bb] crystallization optimization

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 cours

Re: [ccp4bb] crystallization optimization

, 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

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:

Re: [ccp4bb] crystallization optimization

Dear Patrick, Of course this is the best-first choice! I got confused with the names in my previous email. I was reffering to Chen when I mentioned the screens. Kindly, On Wed, Jul 12, 2017 at 12:48 PM, Olga Moroz wrote: > MMS (microseed matrix screening) into several

Re: [ccp4bb] crystallization optimization

MMS (microseed matrix screening) into several screens, as Patrick suggested earlier, would be the first choice for me. Works very often. Good luck! Olga > On 12 Jul 2017, at 08:48, Frank von Delft wrote: > > The point I was failing to make: reducing either

Re: [ccp4bb] crystallization optimization

Vicky, streak seeding is a very good method, but it can be quite a lot of work. Before he tries that, why wouldn't we suggest to Liuqing that he should try MMS - that is, adding a liquid seed stock to random screens? That way he is likely to end up with seeds in wells with similar conditions that

Re: [ccp4bb] crystallization optimization

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

Re: [ccp4bb] crystallization optimization

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

Re: [ccp4bb] crystallization optimization

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

Re: [ccp4bb] crystallization optimization

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 wrote: > > Microseed them into two or

Re: [ccp4bb] crystallization optimization

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

[ccp4bb] crystallization optimization

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