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

[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

[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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[ccp4bb] Tenure-track Faculty positions available at Indiana University School of Medicine

[Takagi, Yuichiro]

Dear Colleagues

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All the best


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Associate Professor
Department of Biochemistry and Molecular Biology
Indiana University School of Medicine
635 Barnhill Drive, Medical Science Building 4003
Indianapolis, IN 46202









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