[ccp4bb] How to replace 2.45M K/Na phosphate buffer - SUMMARY

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: CHOOCH problem
From: Truc Kim curtmik {- at -} GMAIL {- dot -} COM
Date: 2007-07-06

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Subject: How to replace 2.45M K/Na phosphate buffer - SUMMARY
From: Claudia Scotti claudiascotti {- at -} HOTMAIL {- dot -} COM
Date: 2007-07-06

Dear List,

I'd like to thank all of you who answered my question.

All answers except one give suggestions on how to check-improve on the diffraction of the crystals I already have, which is something I didn't hope for.

You can find a summary below.

Thanks again for your precious help,

Claudia

-------------------------------------------------------
Original message:

Dear list,

I've crystals in a condition including 2,45 M K/Na phosphate buffer for the well condition, and 1 microl protein + 0.8 microl Na/K phosphate buffer + 0.25 microl 30% 1,6-hexanediol in the drop condition.

These crystals dissolve in several cryoprotectant solutions and survive only in malonic acid, with which they give diffraction at 7-8 A.

Among the several different possibilities, I'd like to try to optimize the crystals, and one possiblity among others would be to change the buffer.

Any suggestions, please, about how to replace K/Na phosphate? At these concentrations it is acting as a buffer but also as a precipitant and I was wondering if anybody has experienced nice buffer-salt combinations that proved to be useful starting from a similar condition.

Thanks a lot,

Claudia
-------------------------------------------------------

Summary:

1. How to replace K/Na phosphate

Adriana Miele:
Try a combination of ammonium sulphate and sodium sulphate + MES as a buffer. Or: try the Stura
footprint screen that contains other salts beyond phsophate.
---------
By the way: I've already tried this using the QIAgen AmSO4 suite as a guide and home-made solutions, and keeping both 1,6-hexanediol and 0.1 M MES as a buffer. I've now got crystals in several conditions.
Claudia
----------

2. How to check-improve on diffraction

Valerie Biou :
I have had crystals that grew in high concentration phosphate buffer. this was in the old time when data collection was done without cryocooling and the crystals were very temperature sensitive. they grew in the cold room and I had to mount them in a capillary and seal the capillary with glue instead of wax because the wax heat made them dissolve. at this condition, they diffracted to high resolution.
have you tried one or 2 shots at room T ?

James Murray
You do not mention what the room temperature diffraction is like. If you have not tested it already, you should do so - even without malonate, you might only get 7-8A diffraction. Assuming the room-temp diffraction is OK, you could just try increasing the concentration of phosphate buffer - high salt can also act as a cryoprotectant.

Shabir Najmudin
Have you tried using Paratone N as cryouprotectant?

Sergio Martinez Rodriguez
I do not know if somebody has already answered you, but have you tried
directly to diffract your cystal without other cryoprotectant? actually,
1,6-hexanediol can act as a cryoprotectant, so I think that when using
your cryo, you won´t have any ice ring. Apart of that, and as somebody
suggest in the mail list, have you tried RT difracction?

Kornelius Zeth
at around 4 molar P-buffer freezes clean.

Christophe Wirth
After nucleation and crystal growth I would try to increase the level of
salt in the reservoir and seal it again. This will slowly increase the
concentration of the drop. You can do this several times until you reach
a high enough concentration to make it cryo. This may be “softer” then
just soaking the crystal into your cryo solution.

Vaheh Oganesyan:
I've had several cases when crystals did not survive almost cryo protection. In your case you say that crystal diffracts to 7-8 A. Is it possible that this is a limit for that crystal form even with no cryo? Room temp in-house exposure to x-ray will answer this question. So, if your crystal diffracts better at room temp then the cryo agent destroys it. I solved similar problem by adding 5% Glycerol into the crystallization buffer and when crystals grew they allowed addition of Glycerol to 25% (may be more, I did not try).

Jan Abendroth
have you tired oils?

Bernhard Rupp
I wonder why 2.5M phosphate do not suffice as cryoprotectant?
You did try direct mounting?

Richard Baxter
I don't understand why you have to replace the Na/K PO4. If the crystals
dissolve in cryoprotectant isn't that the fault of the cryoprotectant? I
have dissolved common cryoprotectants (glycerol, EG, sucrose, glucose)
in at least 2.3 M Na/K PO4 to 20-30%. Just in case, you are making up a
true solution, not just mixing the resevoir 70:30 with glycerol (in this
case you have reduced the precipitant phosphate concentration and that
may be why the crystals are dissolving).

I did successfully transfer crystals from Na/K PO4 to Na PO4 after they
had grown. You can also vary the pH via the monobasic:dibasic phosphate
ratio in the soak buffer once the crystals have grown to try and
stabilize your crystals at a lower phosphate concentration or in the
presence of the cryoprotectant.

Claudia Scotti
Dipartimento di Medicina Sperimentale
Sezione di Patologia Generale
Universita' di Pavia
Piazza Botta, 10
27100 Pavia
Italia
Tel. 0039 0382 986335/8/1
Facs 0039 0382 303673

----------------------------------------
> Date: Fri, 22 Jun 2007 14:42:00 +0200
> From: claudiascotti@HOTMAIL.COM
> Subject: [ccp4bb] How to replace 2.45M K/Na phosphate buffer - 2
> To: CCP4BB@JISCMAIL.AC.UK
>
> Sorry: forgotten the pH: the crystals grow between pH 6.5 and 6.7.
>
> Claudia
>
>
> Claudia Scotti
> Dipartimento di Medicina Sperimentale
> Sezione di Patologia Generale
> Universita' di Pavia
> Piazza Botta, 10
> 27100 Pavia
> Italia
> Tel. 0039 0382 986335/8/1
> Facs 0039 0382 303673
>
> ----------------------------------------
> > Date: Fri, 22 Jun 2007 14:15:47 +0200
> > From: claudiascotti@HOTMAIL.COM
> > Subject: [ccp4bb] How to replace 2.45M K/Na phosphate buffer
> > To: CCP4BB@JISCMAIL.AC.UK
> >
> > Dear list,
> >
> > I've crystals in a condition including 2,45 M K/Na phosphate buffer for the well condition, and 1 microl protein + 0.8 microl Na/K phosphate buffer + 0.25 microl 30% 1,6-hexanediol in the drop condition.
> >
> > These crystals dissolve in several cryoprotectant solutions and survive only in malonic acid, with which they give diffraction at 7-8 A.
> >
> > Among the several different possibilities, I'd like to try to optimize the crystals, and one possiblity among others would be to change the buffer.
> >
> > Any suggestions, please, about how to replace K/Na phosphate? At these concentrations it is acting as a buffer but also as a precipitant and I was wondering if anybody has experienced nice buffer-salt combinations that proved to be useful starting from a similar condition.
> >
> > Thanks a lot,
> >
> > Claudia
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> > Claudia Scotti
> > Dipartimento di Medicina Sperimentale
> > Sezione di Patologia Generale
> > Universita' di Pavia
> > Piazza Botta, 10
> > 27100 Pavia
> > Italia
> > Tel. 0039 0382 986335/8/1
> > Facs 0039 0382 303673
> > _________________________________________________________________
> > Make every IM count. Download Windows Live Messenger and join the i’m Initiative now. It’s free.
> > http://im.live.com/messenger/im/home/?source=TAGWL_June07
>
> _________________________________________________________________
> Make every IM count. Download Windows Live Messenger and join the i’m Initiative now. It’s free.
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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: CHOOCH problem
From: Truc Kim curtmik {- at -} GMAIL {- dot -} COM
Date: 2007-07-06

Next message:
Subject: Solving structure of protein-protein complexes using MR
From: Joe Smith ewaldxray {- at -} GMAIL {- dot -} COM
Date: 2007-07-06