Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-23 Thread Boniecki, Michal 
In my case what simply worked is to put your solution with 40% glycerol on the 
side of the drop and wait for crystals to stop dancing. Fish them out swiping 
through the side were you have put your solution. Or I have used MPD as 
cryoprotectant and freeze them in cold room (4C) its amazing what cold air 
humidity can do for your drop. No problem with dancing crystals.

Michal Boniecki

On Aug 23, 2018, at 11:38, James Holton 
mailto:jmhol...@slac.stanford.edu>> wrote:


Ahh, yes, the "dancing crystals problem".  The good news is alcohols are really 
good cryoprotectants as well as excellent precipitants.  Shame their use has 
fallen out of favor over the years, but I guess as drop sizes got smaller the 
evaporation problems got worse and worse.

In my humble opinion, this is just an extreme case of a problem we all have 
already.  Evaporation during harvesting is an insidious issue that is hard to 
monitor.  It's not just alcohol, but water that evaporates, and some buffers 
are volatile too (like ammonium and acetate ions).  Volatile buffers mean the 
pH changes over time.  All this can easily lead to non-isomorphism between the 
first crystal you mount vs the last.  An excellent review is: 
https://dx.doi.org/10.1107%2FS1399004714012310

It has already been suggested that you surround your harvesting environment 
with a wet towel (aka Kimwipe) soaked with the well solution, and this is a 
good idea to try and keep the local humidity (or alcohol-idity?) up.  Another 
possibility is to make up 30-40 mL of your well solution, put that into a 50 mL 
conical tube and use one of those stone fish-tank aerators to bubble air or N2 
gas through the appropriate solution for generating just the right 
"atomosphere" that your crystals are used to (see attached photo).  You can 
then direct that gas through an additional length of hose in the general 
direction of your well before you crack it open.  The point is to keep your 
crystals unaware of the fact that they are about to be harvested for as long as 
possible.

In your case you have an excellent assay for when you have kept the harvesting 
environment properly controlled: the crystals will stop dancing.

There are some devices on the market now, such as the "HC1" from Arinax, or the 
"Watershed" from MiTeGen that are a much more sophisticated way to do this, but 
check with the vendor before filling it with alcohol.  Some seals don't like 
non-aqueous liquids.  I expect there may be a safety concern about generating 
large amounts of alcohol vapor, especially isopropanol.  You don't want to 
breathe that in.  Best to keep away from open flames and work in a 
well-ventilated area, like the hood.  Ethanol is less toxic but on a per-capita 
basis more dangerous.  At the very least, don't drive yourself home afterwards.

-James Holton
MAD Scientist


On 8/14/2018 1:58 PM, Thomas Krey wrote:
Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de




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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-23 Thread James Holton 


Ahh, yes, the "dancing crystals problem".  The good news is alcohols are 
really good cryoprotectants as well as excellent precipitants. Shame 
their use has fallen out of favor over the years, but I guess as drop 
sizes got smaller the evaporation problems got worse and worse.


In my humble opinion, this is just an extreme case of a problem we all 
have already.  Evaporation during harvesting is an insidious issue that 
is hard to monitor.  It's not just alcohol, but water that evaporates, 
and some buffers are volatile too (like ammonium and acetate ions).  
Volatile buffers mean the pH changes over time. All this can easily lead 
to non-isomorphism between the first crystal you mount vs the last.  An 
excellent review is: https://dx.doi.org/10.1107%2FS1399004714012310


It has already been suggested that you surround your harvesting 
environment with a wet towel (aka Kimwipe) soaked with the well 
solution, and this is a good idea to try and keep the local humidity (or 
alcohol-idity?) up.  Another possibility is to make up 30-40 mL of your 
well solution, put that into a 50 mL conical tube and use one of those 
stone fish-tank aerators to bubble air or N2 gas through the appropriate 
solution for generating just the right "atomosphere" that your crystals 
are used to (see attached photo). You can then direct that gas through 
an additional length of hose in the general direction of your well 
before you crack it open.  The point is to keep your crystals unaware of 
the fact that they are about to be harvested for as long as possible.


In your case you have an excellent assay for when you have kept the 
harvesting environment properly controlled: the crystals will stop dancing.


There are some devices on the market now, such as the "HC1" from Arinax, 
or the "Watershed" from MiTeGen that are a much more sophisticated way 
to do this, but check with the vendor before filling it with alcohol.  
Some seals don't like non-aqueous liquids.  I expect there may be a 
safety concern about generating large amounts of alcohol vapor, 
especially isopropanol.  You don't want to breathe that in.  Best to 
keep away from open flames and work in a well-ventilated area, like the 
hood.  Ethanol is less toxic but on a per-capita basis more dangerous.  
At the very least, don't drive yourself home afterwards.


-James Holton
MAD Scientist



On 8/14/2018 1:58 PM, Thomas Krey wrote:


Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening 
vapor diffusion experiment in either


15% (v/v) Reagent alcohol

HEPES Na pH 7.5

0.2 M MgCl2

or in

27% Isopropanol

0.18 M MgCl2

90 mM HEPES Na pH 7.5

10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a 
good suggestion to stabilize the swirling movements? Does anyone have 
experience, whether these conditions alone can serve as 
cryo-protectant (i.e., do we really have to fish, move into cryo 
solution and fish again)?


Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas

Prof. Dr. Thomas Krey

Hannover Medical School

Institute of Virology

Structural Virology Group

Carl-Neuberg-Str. 1

D-30625 Hannover

phone: +49 (0) 511 - 532 4308

email: krey.tho...@mh-hannover.de




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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-16 Thread Patrick Shaw Stewart 
Hi Thomas

One problem with conventional approaches is that you tend to get high
mosaicity when harvesting from drops with isopropanol.

A very good method, invented by my old friend Lesley Haire, that has always
worked (so far!) is to use microbatch-under-oil with a plate called the
Vapor Batch plate.

   - Set up the drops with everything in them except for the isopropanol.


   - Cover the drops with 50:50 paraffin and silicone oil ("Al's Oil").


   - Put a few mL of 27% isopropanol in the "moat" around the outside of
   the plate.


The isopropanol will saturate the oil and then diffuse into the drops.  You
can take your time and harvest the crystals through the oil. The oil
protects the crystals, preventing evaporation of isopropanol.

Let me know if you (or anyone) would like some sample plates to try this
out.

See example and ref. below.

Best wishes, Patrick

___


Example: https://www.douglas.co.uk/winner1.htm

*G. B. Mortuza et al.. High-resolution structure of a retroviral capsid
hexameric amino-terminal domain.  Nature 431 (2004), pp 481-485. (This
paper describes the use of the vapor batch plate with isopropanol.)*

Sent from mobile

Patrick Shaw Stewart
+44 7901 548 201

On Wed, 15 Aug 2018, 08:41 Yvonne Thielmann,  wrote:

> Dear Thomas,
>
> maybe you can try to overlay your drop with LV CryoOil from Jena
> Bioscience. Then the evaporation of the solvent is slowed down and the
> crystals are directly cryoprotected when you move the crystals through
> the oil. We had quite good results when we cryoprotected crystals in
> this oil.
>
> Best wishes,
> Yvonne
>
>
> --
> Dr. Yvonne Thielmann
> Max Planck Institute of Biophysics
> Molecular Membrane Biology
> Max-von-Laue-Strasse 3
> 60438 Frankfurt / Main
> Germany
>
> Office +49 69 6303 1056
> Lab +49 69 6303 1074
> Fax +49 69 6303 1002
>
> Am 15.08.2018 um 08:05 schrieb Kajander, Tommi A:
> > Yes sorry, i meant paratone-N also.
> >
> > Tommi
> >
> > Kohteesta: ferrer
> > Lähetetty: keskiviikko 15. elokuuta klo 0.41
> > Aihe: Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
> > Vastaanottaja: ccp4bb@jiscmail.ac.uk
> >
> >
> > Dear Thomas,
> >
> > Alternatively you can try shooting on crystals in the drop, in situ. So
> > fishing, no cryo. But potentially high radiation damage. Can be
> > considered if you have enough crystals, and if your crystallization
> > plate makes it possible.
> >
> > Regards
> >
> > JL
> >
> > On 14/08/2018 20:58, Thomas Krey wrote:
> > Dear crystallization experts,
> >
> > We have 3D protein crystals grown from a microseed matrix screening
> > vapor diffusion experiment in either
> >
> > 15% (v/v) Reagent alcohol
> > HEPES Na pH 7.5
> > 0.2 M MgCl2
> >
> > or in
> >
> > 27% Isopropanol
> > 0.18 M MgCl2
> > 90 mM HEPES Na pH 7.5
> > 10% Glycerol
> >
> > Upon opening the corresponding wells these crystals move quite a bit –
> > presumably due to the volatility of the alcohols. Does anyone have a
> > good suggestion to stabilize the swirling movements? Does anyone have
> > experience, whether these conditions alone can serve as cryo-protectant
> > (i.e., do we really have to fish, move into cryo solution and fish
> again)?
> > Any suggestion or input would be highly welcome.
> >
> > Thank you very much in advance.
> >
> > Thomas
> >
> >
> > Prof. Dr. Thomas Krey
> > Hannover Medical School
> > Institute of Virology
> > Structural Virology Group
> > Carl-Neuberg-Str. 1
> > D-30625 Hannover
> > phone: +49 (0) 511 - 532 4308
> > email: krey.tho...@mh-hannover.de <mailto:krey.tho...@mh-hannover.de>
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
> > --  Jean-Luc Ferrer Institut de
> Biologie
> > Structurale 71 Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 -
> > FRANCE Ph.: +33 (0)4 57 42 85 22 Cell: +33 (0)6 89 45 13 57 email:
> > jean-luc.fer...@ibs.fr <mailto:jean-luc.fer...@ibs.fr>
> > 
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
> >
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
> 
>
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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-15 Thread Yvonne Thielmann 

Dear Thomas,

maybe you can try to overlay your drop with LV CryoOil from Jena 
Bioscience. Then the evaporation of the solvent is slowed down and the 
crystals are directly cryoprotected when you move the crystals through 
the oil. We had quite good results when we cryoprotected crystals in 
this oil.


Best wishes,
Yvonne


--
Dr. Yvonne Thielmann
Max Planck Institute of Biophysics
Molecular Membrane Biology
Max-von-Laue-Strasse 3
60438 Frankfurt / Main
Germany

Office +49 69 6303 1056
Lab +49 69 6303 1074
Fax +49 69 6303 1002

Am 15.08.2018 um 08:05 schrieb Kajander, Tommi A:

Yes sorry, i meant paratone-N also.

Tommi

Kohteesta: ferrer
Lähetetty: keskiviikko 15. elokuuta klo 0.41
Aihe: Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
Vastaanottaja: ccp4bb@jiscmail.ac.uk


Dear Thomas,

Alternatively you can try shooting on crystals in the drop, in situ. So 
fishing, no cryo. But potentially high radiation damage. Can be 
considered if you have enough crystals, and if your crystallization 
plate makes it possible.


Regards

JL

On 14/08/2018 20:58, Thomas Krey wrote:
Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening 
vapor diffusion experiment in either


15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a 
good suggestion to stabilize the swirling movements? Does anyone have 
experience, whether these conditions alone can serve as cryo-protectant 
(i.e., do we really have to fish, move into cryo solution and fish again)?

Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de <mailto:krey.tho...@mh-hannover.de>


To unsubscribe from the CCP4BB list, click the following link:
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--  Jean-Luc Ferrer Institut de Biologie 
Structurale 71 Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 - 
FRANCE Ph.: +33 (0)4 57 42 85 22 Cell: +33 (0)6 89 45 13 57 email: 
jean-luc.fer...@ibs.fr <mailto:jean-luc.fer...@ibs.fr> 


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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-15 Thread Kajander, Tommi A 
Yes sorry, i meant paratone-N also.

Tommi

Kohteesta: ferrer
Lähetetty: keskiviikko 15. elokuuta klo 0.41
Aihe: Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
Vastaanottaja: ccp4bb@jiscmail.ac.uk


Dear Thomas,

Alternatively you can try shooting on crystals in the drop, in situ. So 
fishing, no cryo. But potentially high radiation damage. Can be considered if 
you have enough crystals, and if your crystallization plate makes it possible.

Regards

JL

On 14/08/2018 20:58, Thomas Krey wrote:
Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de<mailto:krey.tho...@mh-hannover.de>


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

--  Jean-Luc Ferrer Institut de Biologie 
Structurale 71 Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 - FRANCE Ph.: 
+33 (0)4 57 42 85 22 Cell: +33 (0)6 89 45 13 57 email: 
jean-luc.fer...@ibs.fr<mailto:jean-luc.fer...@ibs.fr> 

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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread ferrer 

Dear Thomas,

Alternatively you can try shooting on crystals in the drop, in situ. So 
fishing, no cryo. But potentially high radiation damage. Can be 
considered if you have enough crystals, and if your crystallization 
plate makes it possible.


Regards

JL

On 14/08/2018 20:58, Thomas Krey wrote:


Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening 
vapor diffusion experiment in either


15% (v/v) Reagent alcohol

HEPES Na pH 7.5

0.2 M MgCl2

or in

27% Isopropanol

0.18 M MgCl2

90 mM HEPES Na pH 7.5

10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a 
good suggestion to stabilize the swirling movements? Does anyone have 
experience, whether these conditions alone can serve as 
cryo-protectant (i.e., do we really have to fish, move into cryo 
solution and fish again)?


Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas

Prof. Dr. Thomas Krey

Hannover Medical School

Institute of Virology

Structural Virology Group

Carl-Neuberg-Str. 1

D-30625 Hannover

phone: +49 (0) 511 - 532 4308

email: krey.tho...@mh-hannover.de




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



--

Jean-Luc Ferrer
Institut de Biologie Structurale
71 Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9 - FRANCE

Ph.:  +33 (0)4 57 42 85 22
Cell: +33 (0)6 89 45 13 57
email: jean-luc.fer...@ibs.fr





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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Kevin Jin 
I just give an example of oil. Indeed, paraffin is also a very good option.
In some cases (<=5% alcohol).  Here is the difference I observed between
paraffin and paratone oil.
1. Paraffin oil has low viscosity. Paratone oil is too sticky with high
viscosity. Sometimes, I mix them with different ratio.
2. In the crystallization with high concentration of alcohol (>10%), I
prefer paratone oil, which can form a bulky cover immediately. Paraffin oil
may spread out and could not cover the drop completely.

The basic idea is to proved an effective shell to prevent solvent
evaporation.



On Tue, Aug 14, 2018 at 12:56 PM Patrick Loll  wrote:

> I second (third?) what Tommi and Kevin said about using an oil to cover
> the drop to slow evaporation (I like paraffin for this—not too viscous).
> Here’s an additional nuance: Saturate the oil with the alcohol first,
> before using it to cover the drop.
>
> > On 14 Aug 2018, at 2:58 PM, Thomas Krey 
> wrote:
> >
> > Dear crystallization experts,
> >
> > We have 3D protein crystals grown from a microseed matrix screening
> vapor diffusion experiment in either
> >
> > 15% (v/v) Reagent alcohol
> > HEPES Na pH 7.5
> > 0.2 M MgCl2
> >
> > or in
> >
> > 27% Isopropanol
> > 0.18 M MgCl2
> > 90 mM HEPES Na pH 7.5
> > 10% Glycerol
> >
> > Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
> > Any suggestion or input would be highly welcome.
> >
> > Thank you very much in advance.
> >
> > Thomas
> >
> >
> > Prof. Dr. Thomas Krey
> > Hannover Medical School
> > Institute of Virology
> > Structural Virology Group
> > Carl-Neuberg-Str. 1
> > D-30625 Hannover
> > phone: +49 (0) 511 - 532 4308
> > email: krey.tho...@mh-hannover.de
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
>
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Patrick Loll 
I second (third?) what Tommi and Kevin said about using an oil to cover the 
drop to slow evaporation (I like paraffin for this—not too viscous). Here’s an 
additional nuance: Saturate the oil with the alcohol first, before using it to 
cover the drop. 

> On 14 Aug 2018, at 2:58 PM, Thomas Krey  wrote:
> 
> Dear crystallization experts,
>  
> We have 3D protein crystals grown from a microseed matrix screening vapor 
> diffusion experiment in either
>  
> 15% (v/v) Reagent alcohol
> HEPES Na pH 7.5
> 0.2 M MgCl2 
>  
> or in 
>  
> 27% Isopropanol
> 0.18 M MgCl2
> 90 mM HEPES Na pH 7.5
> 10% Glycerol
>  
> Upon opening the corresponding wells these crystals move quite a bit – 
> presumably due to the volatility of the alcohols. Does anyone have a good 
> suggestion to stabilize the swirling movements? Does anyone have experience, 
> whether these conditions alone can serve as cryo-protectant (i.e., do we 
> really have to fish, move into cryo solution and fish again)? 
> Any suggestion or input would be highly welcome.
>  
> Thank you very much in advance.
>  
> Thomas
>  
>  
> Prof. Dr. Thomas Krey
> Hannover Medical School  
> Institute of Virology
> Structural Virology Group
> Carl-Neuberg-Str. 1 
> D-30625 Hannover
> phone: +49 (0) 511 - 532 4308
> email: krey.tho...@mh-hannover.de
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu



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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Kevin Jin 
Hi Thomas,

This is usual when high volatile solvent is used in crystallization
(membrane or glycoproteins). The crystal may looks very nice with sharp
edges. When you open the cover glass, you may see a very thin film formed
on the hang-on drops. Once you touch the drop, then crystals move very
quickly like flying and start cracking.

Here is what you may try:
1. When you open the cover glass, using a big drop of Paratone oil to cover
(wrap) the drop immediately and completely.
2. Then, inject the cryoprotectant into the drop wrapped by paratone oil.
3.  When you wash the crystal in cryoprotectant and fish the crystal out,
please always keep all of the operation in a large paratone oil drop.
4. Make sure there is always a layer of paratone oil on the loop when you
freeze it in LN2.

I hope this will be helpful.

Regards,

Kevin





On Tue, Aug 14, 2018 at 12:00 PM Thomas Krey 
wrote:

> Dear crystallization experts,
>
>
>
> We have 3D protein crystals grown from a microseed matrix screening vapor
> diffusion experiment in either
>
>
>
> 15% (v/v) Reagent alcohol
>
> HEPES Na pH 7.5
>
> 0.2 M MgCl2
>
>
>
> or in
>
>
>
> 27% Isopropanol
>
> 0.18 M MgCl2
>
> 90 mM HEPES Na pH 7.5
>
> 10% Glycerol
>
>
>
> Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
>
> Any suggestion or input would be highly welcome.
>
>
>
> Thank you very much in advance.
>
>
>
> Thomas
>
>
>
>
>
> Prof. Dr. Thomas Krey
>
> Hannover Medical School
>
> Institute of Virology
>
> Structural Virology Group
>
> Carl-Neuberg-Str. 1
>
> D-30625 Hannover
>
> phone: +49 (0) 511 - 532 4308
>
> email: krey.tho...@mh-hannover.de
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Petri Kursula 
Hi,
you could try picking in the cold room. Provided the temperature change does 
not kill the crystals, this sometimes worked fine for me in similar cases.
Petri


Petri Kursula
--
Professor 
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula 

petri.kurs...@uib.no 
--
Group Leader, Adjunct Professor
Faculty of Biochemistry and Molecular Medicine
University of Oulu, Finland
--



> On 14 Aug 2018, at 20:58, Thomas Krey  wrote:
> 
> Dear crystallization experts,
>  
> We have 3D protein crystals grown from a microseed matrix screening vapor 
> diffusion experiment in either
>  
> 15% (v/v) Reagent alcohol
> HEPES Na pH 7.5
> 0.2 M MgCl2 
>  
> or in 
>  
> 27% Isopropanol
> 0.18 M MgCl2
> 90 mM HEPES Na pH 7.5
> 10% Glycerol
>  
> Upon opening the corresponding wells these crystals move quite a bit – 
> presumably due to the volatility of the alcohols. Does anyone have a good 
> suggestion to stabilize the swirling movements? Does anyone have experience, 
> whether these conditions alone can serve as cryo-protectant (i.e., do we 
> really have to fish, move into cryo solution and fish again)? 
> Any suggestion or input would be highly welcome.
>  
> Thank you very much in advance.
>  
> Thomas
>  
>  
> Prof. Dr. Thomas Krey
> Hannover Medical School  
> Institute of Virology
> Structural Virology Group
> Carl-Neuberg-Str. 1 
> D-30625 Hannover
> phone: +49 (0) 511 - 532 4308
> email: krey.tho...@mh-hannover.de 
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Kajander, Tommi A 
Hi, One way is to cover the drop with e.g. paraffin oil (to prevent 
evaporation) and fish it out in the oil. Have had luck with that in some cases.

Tommi

On 14 Aug 2018, at 22:09, Bonsor, Daniel 
mailto:dbon...@som.umaryland.edu>> wrote:

I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir 
to the drop. The larger volume of the drop appears to slow down the whizzing of 
the crystals and allows you to get a few crystals. Though it still occurs. You 
could also cool the area down or move into the cold room if your crystals 
survive the transfer as evaporation should be less at 4oC.

You can also spot several 1-2uL of neat reservoir solution around the drop to 
create a local vapor barrier to prevent evaporation of the drop. It can 
completely stop the movement of crystals for a few minutes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas 
Krey
Sent: Tuesday, August 14, 2018 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@jiscmail.ac.uk>
Subject: [ccp4bb] Fishing crystals from volatile solvent as precipitant

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email 
system. Whether the sender is known or not known, hover over any links before 
clicking and use caution opening attachments.

Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de<mailto:krey.tho...@mh-hannover.de>




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Tommi Kajander, Ph.D., PI
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1 (P.O. Box 65)
00014 Helsinki, Finland
p. +358-2-941-58904 / +358-050-4480991
tommi.kajan...@helsinki.fi<mailto:tommi.kajan...@helsinki.fi>
http://www.biocenter.helsinki.fi/bi/kajander/






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Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Bonsor, Daniel 
I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir 
to the drop. The larger volume of the drop appears to slow down the whizzing of 
the crystals and allows you to get a few crystals. Though it still occurs. You 
could also cool the area down or move into the cold room if your crystals 
survive the transfer as evaporation should be less at 4oC.

You can also spot several 1-2uL of neat reservoir solution around the drop to 
create a local vapor barrier to prevent evaporation of the drop. It can 
completely stop the movement of crystals for a few minutes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas 
Krey
Sent: Tuesday, August 14, 2018 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fishing crystals from volatile solvent as precipitant

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email 
system. Whether the sender is known or not known, hover over any links before 
clicking and use caution opening attachments.

Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de<mailto:krey.tho...@mh-hannover.de>




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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[ccp4bb] Fishing crystals from volatile solvent as precipitant

2018-08-14 Thread Thomas Krey 
Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de




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https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1