[ccp4bb] Test of cryoconditions without X-rays or cryostream

[Claudia Scotti]

 

Dear List,

 

Sorry for the probably silly question.

 

Any suggestions to test cryoconditions without X-rays or cryostream? 

 

I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is it 
enough to try to freeze the cryoconditions in liquid nitrogen checking under 
the microscope (or by eye) or is this still risky?

 

Thanks,

 

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



_
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Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Karthik S]

I have not heard of anyone checking for suitable cryo in that fashion how
fast can you do it before it is affected by room temp and how would you know
about the ice rings, but when you have crystal(s) and not crystal you can
freeze different ones in several different cryos (sugars, glycerol etc) and
take them with you to the trip. good luck!
--
Karthik

Graduate Student-Biophysics
University of Michigan
Ann Arbor, MI 48109
karth...@umich.edu
734-763-3384

On Tue, Sep 22, 2009 at 4:24 AM, Claudia Scotti
wrote:

>
> Dear List,
>
> Sorry for the probably silly question.
>
> Any suggestions to test cryoconditions without X-rays or cryostream?
>
> I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is
> it enough to try to freeze the cryoconditions in liquid nitrogen checking
> under the microscope (or by eye) or is this still risky?
>
> Thanks,
>
> 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
>
>
>
> --
> With Windows Live, you can organize, edit, and share your 
> photos.
>

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[mesters]

Dear Claudia,

please be so kind and tell us all what is / are the crystallization 
condition(s)? It would help to us to answer your question!


- J. -



Claudia Scotti wrote:
 
Dear List,
 
Sorry for the probably silly question.
 
Any suggestions to test cryoconditions without X-rays or cryostream?
 
I'd need to freeze crystals before going to ESRF and I'm a bit 
anxious. Is it enough to try to freeze the cryoconditions in liquid 
nitrogen checking under the microscope (or by eye) or is this still risky?
 
Thanks,
 
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





With Windows Live, you can organize, edit, and share your photos. 




--
Dr. Jeroen R. Mesters
Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
Institut für Biochemie, Universität zu Lübeck
Zentrum für Medizinische Struktur- und Zellbiologie
Ratzeburger Allee 160, D-23538 Lübeck
Tel: +49-451-5004065, Fax: +49-451-5004068
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.selfish-brain.org
Http://www.opticryst.org
--
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which grain will grow and which will not - speak then to me  (Macbeth)
--

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Frank von Delft]

Hi Claudia

"By eye" is not a bad approximation.  The trick I learnt at the JCSG (I 
forget the originator, sorry) is to suck up some solution in a P2 
pipette tip (the clear ones), dip it in lN2, and then hold it just above 
the lN2 (where it's still cold) to check for clearness or not.


Is a good predictor especially if your loops are very small (volume is a 
big factor in whether you can achieve vitrification or not).


Cheers
phx



On Tue, Sep 22, 2009 at 4:24 AM, Claudia Scotti 
mailto:claudiasco...@hotmail.com>> wrote:


 
Dear List,
 
Sorry for the probably silly question.
 
Any suggestions to test cryoconditions without X-rays or cryostream?
 
I'd need to freeze crystals before going to ESRF and I'm a bit

anxious. Is it enough to try to freeze the cryoconditions in
liquid nitrogen checking under the microscope (or by eye) or is
this still risky?
 
Thanks,
 
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




With Windows Live, you can organize, edit, and share your photos.

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Arthur Glasfeld]

It might be risky (and silly), but I have done this successfully.  I  
just put a small dewar of liquid N2 under the scope, focussed  
directly above the surface and looked to see if cryo solutions froze  
clear or not.  The ones that did worked out when we got to the  
beamline (we'd verified that the crystals survived the treatment in  
the solutions by eye as well).  Only tried it that once, so I have no  
idea how reliable it is.


Good luck,

Arthur Glasfeld
Department of Chemistry
Reed College
3203 SE Woodstock Blvd.
Portland, OR 97202
USA


On Sep 22, 2009, at 1:24 AM, Claudia Scotti wrote:



Dear List,

Sorry for the probably silly question.

Any suggestions to test cryoconditions without X-rays or cryostream?

I'd need to freeze crystals before going to ESRF and I'm a bit  
anxious. Is it enough to try to freeze the cryoconditions in liquid  
nitrogen checking under the microscope (or by eye) or is this still  
risky?


Thanks,

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




With Windows Live, you can organize, edit, and share your photos.

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Paul Leonard]

Hi Claudia

It really is best to test the cryo condition in an X-ray beam.

However, if you don't have access to X-rays in house you could look at the
hampton crystallization screen conditions (or another supplier).  Find a
condition with a similar amount and type of precipitant as you have in
your crystallisation condition and look up how much glycerol they added as
cryoprotectant.  Then try making up your own crystallisation condition
with the amount of glycerol they recommend and freeze your crystals in
that solution.

Also try crystal soaks of varying lengths of time (30 seconds - 5 min) in
the cryo condition as well as trying to soak some crystals where you
gradually increase the %glycerol in the crystal soak prior to freezing (in
2% glycerol steps for example).

see
http://hamptonresearch.com/product_detail.aspx?cid=1&sid=20&pid=3

Good luck.

Paul
>
>
>
> Dear List,
>
>
>
> Sorry for the probably silly question.
>
>
>
> Any suggestions to test cryoconditions without X-rays or cryostream?
>
>
>
> I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is
> it enough to try to freeze the cryoconditions in liquid nitrogen checking
> under the microscope (or by eye) or is this still risky?
>
>
>
> Thanks,
>
>
>
> 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
>
>
>
> _
> With Windows Live, you can organize, edit, and share your photos.
> http://www.microsoft.com/middleeast/windows/windowslive/products/photo-gallery-edit.aspx


Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Elspeth Garman]

or see concentrations given in:
McFerrin and Snell
J.Appl.Cryst (2002) 35, 538
and
Mitchell and Garman,
J.Appl.Cryst. (1996) 29, 584
Good luck
EFG



Paul Leonard wrote:

Hi Claudia

It really is best to test the cryo condition in an X-ray beam.

However, if you don't have access to X-rays in house you could look at the
hampton crystallization screen conditions (or another supplier).  Find a
condition with a similar amount and type of precipitant as you have in
your crystallisation condition and look up how much glycerol they added as
cryoprotectant.  Then try making up your own crystallisation condition
with the amount of glycerol they recommend and freeze your crystals in
that solution.

Also try crystal soaks of varying lengths of time (30 seconds - 5 min) in
the cryo condition as well as trying to soak some crystals where you
gradually increase the %glycerol in the crystal soak prior to freezing (in
2% glycerol steps for example).

see
http://hamptonresearch.com/product_detail.aspx?cid=1&sid=20&pid=3

Good luck.

Paul
  


Dear List,



Sorry for the probably silly question.



Any suggestions to test cryoconditions without X-rays or cryostream?



I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is
it enough to try to freeze the cryoconditions in liquid nitrogen checking
under the microscope (or by eye) or is this still risky?



Thanks,



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



_
With Windows Live, you can organize, edit, and share your photos.
http://www.microsoft.com/middleeast/windows/windowslive/products/photo-gallery-edit.aspx




Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu
  


--
**NOTE NEW E-MAIL ADDRESS AND PHONE NUMBER

 Elspeth F. Garman,
 President, British Crystallographic Association
 Professor of Molecular Biophysics, University of Oxford
 Visiting Professor in Chemistry, University of Durham
 Postal address:
 Laboratory of Molecular Biophysics,
 Department of Biochemistry,
 University of Oxford,  Tel: (44)-1865-613297
 South Parks Road,  FAX: (44)-1865-613201
 OXFORD, OX1 3QU, U.K. E-mail: elspeth.gar...@bioch.ox.ac.uk
http://www.biop.ox.ac.uk/www/garman/gindex.html 
-

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Jürgen Bosch]

I think I posted this link maybe two years ago:
http://idb.exst.jaxa.jp/db_data/protein/search-e.php?

Jürgen

On Sep 22, 2009, at 12:48 PM, Elspeth Garman wrote:


or see concentrations given in:
McFerrin and Snell
J.Appl.Cryst (2002) 35, 538
and
Mitchell and Garman,
J.Appl.Cryst. (1996) 29, 584
Good luck
EFG



Paul Leonard wrote:

Hi Claudia

It really is best to test the cryo condition in an X-ray beam.

However, if you don't have access to X-rays in house you could look  
at the
hampton crystallization screen conditions (or another supplier).   
Find a
condition with a similar amount and type of precipitant as you have  
in
your crystallisation condition and look up how much glycerol they  
added as
cryoprotectant.  Then try making up your own crystallisation  
condition
with the amount of glycerol they recommend and freeze your crystals  
in

that solution.

Also try crystal soaks of varying lengths of time (30 seconds - 5  
min) in

the cryo condition as well as trying to soak some crystals where you
gradually increase the %glycerol in the crystal soak prior to  
freezing (in

2% glycerol steps for example).

see
http://hamptonresearch.com/product_detail.aspx?cid=1&sid=20&pid=3

Good luck.

Paul



Dear List,



Sorry for the probably silly question.



Any suggestions to test cryoconditions without X-rays or cryostream?



I'd need to freeze crystals before going to ESRF and I'm a bit  
anxious. Is
it enough to try to freeze the cryoconditions in liquid nitrogen  
checking

under the microscope (or by eye) or is this still risky?



Thanks,



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



_
With Windows Live, you can organize, edit, and share your photos.
http://www.microsoft.com/middleeast/windows/windowslive/products/photo-gallery-edit.aspx




Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu



--
**NOTE NEW E-MAIL ADDRESS AND PHONE NUMBER

 Elspeth F. Garman,
 President, British Crystallographic Association
 Professor of Molecular Biophysics, University of Oxford
 Visiting Professor in Chemistry, University of Durham
 Postal address:
 Laboratory of Molecular Biophysics,
 Department of Biochemistry,
 University of Oxford,  Tel: (44)-1865-613297
 South Parks Road,  FAX: (44)-1865-613201
 OXFORD, OX1 3QU, U.K. E-mail: elspeth.gar...@bioch.ox.ac.uk
http://www.biop.ox.ac.uk/www/garman/gindex.html
-


-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Clemens Grimm]

Checking Cryo-Conditions under the microscope can actually be very  
useful. Even if you have access to a X-ray beam, simple pre-tests can  
be very informative.


This can be done as follows:

A 24well plate cover is placed on a stereo microscope open side down,  
a second one on top of it the other way around. Carefully pour lN2  
into it (might make a crackling noise, however the plastic should  
stand it). A 1 mm loop is placed on top of a 'crystal wand' tool,  
dipped first into the cryo buffer under investigation and then quickly  
into the lN2 on the microscope. Hold the loop right under the lN2  
surface and focus. Vitrification of the shock-cooled buffer can now be  
assesed.
I usually check  a series with increasing concentrations of  
cryo-protectant to determine the critical point for successful  
vitrification. To the final buffer I then add a certain safety margin  
to avoid unpleasant surprises during the synchrotron trip, if an X-ray  
test is not possible.


Clemens


Zitat von Karthik S :


I have not heard of anyone checking for suitable cryo in that fashion how
fast can you do it before it is affected by room temp and how would you know
about the ice rings, but when you have crystal(s) and not crystal you can
freeze different ones in several different cryos (sugars, glycerol etc) and
take them with you to the trip. good luck!
--
Karthik

Graduate Student-Biophysics
University of Michigan
Ann Arbor, MI 48109
karth...@umich.edu
734-763-3384

On Tue, Sep 22, 2009 at 4:24 AM, Claudia Scotti
wrote:



Dear List,

Sorry for the probably silly question.

Any suggestions to test cryoconditions without X-rays or cryostream?

I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is
it enough to try to freeze the cryoconditions in liquid nitrogen checking
under the microscope (or by eye) or is this still risky?

Thanks,

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



--
With Windows Live, you can organize, edit, and share your  
photos.

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[James Holton]

You can put your "dogbowl dewar" under the microscope and focus it on 
the surface of the liquid nitrogen.  This works very well once you have 
got the lighting right.  You want to shine the light on the bottom of 
the dewar so that it acts as a backlight for your loop.  You can hold 
the loop just above the surface of the liquid and it will still be very 
cold, but much easier to see.


However, as has been mentioned there is no substitute for diffraction.  
Transparency of the drop is neither necessary nor sufficient for good 
crystal diffraction.  Salty cryos do cool as amorphous solids, but often 
have a "mottled" surface that is not optically clear, and 
nano-crystalline cubic ice is optically clear, but still gives sharp ice 
rings. 

That said, some protein crystals diffract better in cubic ice than they 
do in amorphous ice.  The trick to the whole thing is avoiding strain in 
the protein crystal lattice because if it strains as it cools then none 
of your unit cells will be exactly the same size, and high-order Fourier 
terms vanish.  It is easy to imagine how strain could build up if your 
solvent channels contract differently than the protein lattice.  The 
problem is that there is no way to know ahead of time how your protein 
crystal lattice will interact with a particular cryo, so you have to 
screen.  I recommend taking a large VARIETY of cryo conditions to the 
beamline for your first trip (don't just freeze everything in 
glycerol).  Mixed cryos are easier to glassify than single-component 
cryos (the "confusion effect").  Don't forget oil, it works about half 
the time (in my experience), and mind the level of liquid nitrogen in 
the dewar or your cooling rate will not be reproducible (Warkentin et 
al., JACr 2006).  Or, better yet, if at all possible take uncooled 
crystals to the beam and learn as you go.  Protein crystals are a lot 
like children, they are all special and unique and expect to be treated 
that way, even after they are fully grown.


-James Holton
MAD Scientist


Claudia Scotti wrote:
 
Dear List,
 
Sorry for the probably silly question.
 
Any suggestions to test cryoconditions without X-rays or cryostream?
 
I'd need to freeze crystals before going to ESRF and I'm a bit 
anxious. Is it enough to try to freeze the cryoconditions in liquid 
nitrogen checking under the microscope (or by eye) or is this still risky?
 
Thanks,
 
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





With Windows Live, you can organize, edit, and share your photos.