Re: [ccp4bb] crystallization optimization

2017-07-13 Thread Patrick Shaw Stewart 
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=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-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwl

Re: [ccp4bb] crystallization optimization

2017-07-13 Thread Keller, Jacob 
I’d be curious whether anyone has ever published an empirical phase diagram 
that looks like the one posted here, since I think real experiments have a lot 
more free parameters than those included in the phase diagram.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf 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 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<mailto: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=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=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 L

Re: [ccp4bb] crystallization optimization

2017-07-13 Thread Oganesyan, Vaheh 
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=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=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:

Re: [ccp4bb] crystallization optimization

2017-07-13 Thread Anthony Savill 
And the Molecular Dimension s 3D screen.

Tony Savill
Molecular Dimensions Ltd.
Unit 6 Goodwin Business Park
Willie Snaith Road
Newmarket, Suffolk.
UK CB8 7SQ

Tel: +44 1638 561051
Fax: +44 1638 660674

Registered Office
Salisbury House
Station Road
Cambridge
CB1 2LA

Registered in England 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 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 
<alun.co...@ucl.ac.uk<mailto:alun.co...@ucl.ac.uk>> wrote:

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 12/07/17 16:54, Frank von Delft wrote:

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 wishes

Patrick











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
 Tel: 44 (0) 148-864-9090<tel:01488%20649090>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 67

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Roger Rowlett 
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.

Cheers,

Roger Rowlett


On Jul 12, 2017 12:01 PM, "Alun R Coker"  wrote:

> 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 12/07/17 16:54, Frank von Delft wrote:
>
> 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 wishes
>
> Patrick
>
>
>
>
>
>
>
>
>
>
>
> On 12 July 2017 at 14:40, Alun R Coker  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.ukDouglas 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 <01488%20649090>US toll-free 1-877-225-2034
> <%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 <020%207679%206703>
> Web: www.ucl.ac.uk/pxmed
>
>
>
>
> --
>  patr...@douglas.co.ukDouglas 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 <01488%20649090>US toll-free 1-877-225-2034
> <(877)%20225-2034>
>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>
>
>
> --
> Dr Alun R. Coker
> Senior

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Debanu Das 
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:
> 
> 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 12/07/17 16:54, Frank von Delft wrote:
>> 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 wishes
>>> 
>>> Patrick
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> On 12 July 2017 at 14:40, Alun R Coker  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 
>  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 
>>  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.ukDouglas 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) 148-864-9090US 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

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Colbert, Christopher 
It’s been my anecdotal experience that the phase diagram for proteins is 
nowhere near as uniform as is presented in most textbooks.  So while in the 
presented diagram, I would definitely support the proposed experiment, knowing 
you are in that region of the phase diagram is many many many experiments.

That being said, crystallization is stochastic, so the more things you do, the 
better off you will be.

Cheers,

Chris
—
Christopher L. Colbert, Ph.D.
Associate Professor
Department of Chemistry and Biochemistry
North Dakota State University
P.O. Box 6050 Dept. 2710
Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Frank von Delft 
<frank.vonde...@sgc.ox.ac.uk<mailto:frank.vonde...@sgc.ox.ac.uk>>
Reply-To: Frank von Delft 
<frank.vonde...@sgc.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.  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 wishes

Patrick











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
 Tel: 44 (0) 148-864-9090<tel:01488%20649090>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<te

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Alun R Coker 
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 12/07/17 16:54, Frank von Delft wrote:


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 wishes

Patrick











On 12 July 2017 at 14:40, Alun R Coker > 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
> 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
> 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
 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 




--
patr...@douglas.co.uk    Douglas 
Instruments Ltd.

 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Frank von Delft 
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 wishes

Patrick











On 12 July 2017 at 14:40, Alun R Coker > 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
> 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
> 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
 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 




--
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

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Evans, Nicola 
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

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Emmanuel Saridakis 
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) D 50 , 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: 

BQ_BEGIN

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 < [ 
mailto:frank.vonde...@sgc.ox.ac.uk | frank.vonde...@sgc.ox.ac.uk ] > wrote: 

BQ_BEGIN



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: 

BQ_BEGIN

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 < [ 
mailto:patr...@douglas.co.uk | patr...@douglas.co.uk ] > wrote: 

BQ_BEGIN


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 < [ mailto:519198...@163.com | 
519198...@163.com ] > wrote: 

BQ_BEGIN
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 






-- 
[ mailto:patr...@douglas.co.uk | 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/ | http://www.douglas.co.uk ] 
Tel: 44 (0) 148-864-9090 US toll-free [ tel:%28877%29%20225-2034 | 
1-877-225-2034 ] 
Regd. England 2177994, VAT Reg. GB 480 7371 36 

BQ_END



BQ_END


BQ_END



BQ_END


BQ_END

-- 
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: [ http://www.ucl.ac.uk/pxmed | www.ucl.ac.uk/pxmed ]

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Alun R Coker 

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 
> 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
> 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
 Tel: 44 (0) 148-864-9090US 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

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Vicky Tsirkone 
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 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 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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034
>>> <%28877%29%20225-2034>
>>>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>>>
>>
>>
>>
>
>
>

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Olga Moroz 
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 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 
>> > 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 
>>> > 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 
>>>  Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 
>>> 
>>>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>>> 
>> 
>> 
>

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Patrick Shaw Stewart 
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 happen to be in the metastable zone of the phase diagram, and also
with seeds in *completely unrelated conditions *that are also in the
metastable zone.  That way you can cast your net very wide.

One simple experiment does so much - it is also an additive experiment : )
 Part of the art of crystallization is to try lots of things without
thinking too much.

Patrick




On 12 July 2017 at 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.ukDouglas 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 <01488%20649090>US toll-free
>>> 1-877-225-2034 <%28877%29%20225-2034>
>>>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>>>
>>
>>
>>
>


-- 
 patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Frank von Delft 
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 
> 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
> 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
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034

 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Vicky Tsirkone 
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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034
>> <%28877%29%20225-2034>
>>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>>
>
>
>

Re: [ccp4bb] crystallization optimization

2017-07-11 Thread Frank von Delft 
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 
> 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
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034

 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] crystallization optimization

2017-07-11 Thread Vicky Tsirkone 
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 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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034
> <(877)%20225-2034>
>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>

Re: [ccp4bb] crystallization optimization

2017-07-10 Thread Patrick Shaw Stewart 
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.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

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[ccp4bb] crystallization optimization

2017-07-10 Thread Liuqing Chen 
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