cryo protection

From: Leonard Thomas
Date: 26 October 2011 17:46


Hi All,

I have run into a very sensitive crystals system when it comes to cryo protecting them.  I have run through the usual suspects and trays are going to be setup with a cryo protectant as part of crystallization cocktail.  The one problem that  seems to be occurring is that the crystals crack as soon as they are transfered out of the original drop.  I am running out of ideas and really would love some new ones.

Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

Dear Len
This is a classic sign of osmotic shock. You can try matching the osmotic pressure of the mother liquor and the cryoprotectant buffer.
For a protocol see Acta Cryst D (1999) 55, 1649 section 6.4
Good luck
Best wishes
Elspeth

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From: Mark J van Raaij 

you may have thought of this already, but you could try cryoprotection in the drop itself.
i.e. slowly adding cryoprotectant to the reservoir, or replacing the reservoir bit by bit with solution containing cryoprotectant, and then adding small volumes to the side of the drop
- for example, exchanging 20% volume cryo-solution with the reservoir, letting equilibrate with the unchanged drop for a few hours, then add 20% volume to the drop from the reservoir (i.e. 0.2 ul if the drop is 1 ul), then add another 20% of cryo to the reservoir, equilibrate a few hrs, etc. - the idea being to very slowly change the drop conditions and minimise risk of cracking.
Of course, you may need patience and many drops of crystals, not just many crystals in a few drops, until you find the cryoprotectant where the crystals do not crack and still diffract, if you are successful at all...
but if it works, you can just harvest from the equilibrated drop and directly flash-cool

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain

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From: Tim Gruene



Dear Len,

just to be on the safe side, my list of 'usual suspects' includes
- - glycerol/PEG400
- - LiCl et al at high concentration
- - Butanediol
- - sugars (glucose/ fructose)
- - oil
- - NaMalonate
- - MPD
...

you mention cracking upon transferring the crystal.
- - do you use a pipet for transfer?
- - addition of cryo TO the drop?
- - did you try slow (several minutes - 1hr) / quick addition of cryo
protectant
- - seeding into slightly different conditions/additive screens
- - seeding into cryo conditions
...

How about collecting data at room temperature?

Hope this list contains some new ideas.

Best wishes,
Tim

- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen



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From: Roger Rowlett

Len,

We have run into this problem from time to time, and it is very frustrating. Here are some things to try, some of which you may have done already:

1. Grow crystals in a small percentage of the cryoprotectant (e.g., 5-10% glycerol). This often allows crystal transfer into a cryo drop without cracking. (Almost never works for us, though.)
2. Do your crystal transfers in the cold room. This slows evaporation markedly, and may prevent crystal cracking. (This works for us some of the time.)
3. Transfer your crystals to gradually higher cryoprotectant concentrations (e.g., to 15% glycerol, then 30% glycerol). (Fiddly, and the crystals get handled a lot, but often works.)
   
4. Use different cryoprotectants. We almost always have fewer cracking issues with glucose than glycerol, but YMMV.
   
5. Avoid transferring the crystal from the drop at all. Just add cryoprotectant to the drop. Even better, add cryoprotectant to the drop gradually, while keeping the drop humidified over well solution. This is our "No-fail" method (this is usually, but not always successful):

http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals#No_fail_cryoprotection

We typically use glucose in this method, but in principle you could try glycerol, MPD, PEG-400, or sodium formate, etc.

Otherwise, you can try to grow out of a cryo condition that doesn't need extra cryoprotectant (been there done that) or give up and shoot at room temp in-house.

Cheers,

_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

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From: Filip Van Petegem


Hello Leonard,

one thing to test is whether transferring your crystals to a drop containing simply well solution also causes cracking. If yes, then the possibility exists that the absence of protein in solution is causing the trouble. In that case, you can transfer the crystals to oil:  you'll be transferring the solution (with protein) in which the crystal grew as well, and slowly remove it without adding anything 'different'.  However, if your crystals crack simply because they are mechanically fragile, then the oil may actually be worse.

Filip

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

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From: Zhou, Tongqing (NIH/VRC) [E]

You can also try to crosslink before transferring to cryo.

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From: Muhammed bashir Khan


Hi Len;

I was having exactly the same problem with my crystals, but when we grow
the crystals in presence of increasing concentration of Glycerol and MPD
starting from 0.5 to 10%. The crystal doesn't appear after 3% of Glycerol
or MPD but the one which appear in 2.5 to 3 % were much resistant to
cracking than the original crystals.

 Good luck

Bashir

--
Muhammad Bashir Khan
**************************************************
Department for Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Austria



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From: Andrew Purkiss-Trew


Another possibility (other than those already mentioned) is to try freezing without a cryoprotectant, by fishing the crystals out onto a mesh and removing all the mother liquor.

The following paper has some details:
"Direct cryocooling of naked crystals: are cryoprotection agents always necessary?"

Erika Pellegrini, Dario Pianoa, and Matthew W. Bowlera

Acta Cryst. (2011). D67, 902–906

--
Andrew Purkiss
X-ray Laboratory Manager
Cancer Research UK
London Research Institute.

----------------------------------------------------------------

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From: Leonard Thomas


A good number of things to try.  Just a little more info that was asked for.  The crystals are grown in Peg 3350 over a range of pH values using Bis-Tris Propane.  The are coming out of 2 different salt conditions.   My feeling is it is an osmolality problem though I also observed cracking when going into a separately made well solution.  I will look it trying a number of suggestions given.

Cheers,

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From: harkewal singh


Len,
May be you have already done this. I would closely check my
crystallization conditions and also check the pH of the cryo. In some
cases, during cryoprotection the pH of the original drop may drastically
different than the cryo solution. Also, sometime back, we were exploring
different cryoprotectant conditions for sensitive crystal and came
across this -
http://www.xtals.org/crystal_cryo.pdf by Artem.

HTH
Harkewal

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From: Mathews, Irimpan I.


One small point:

Just make sure that you are not too off from the contents of the protein solution.  Sometimes protein solution may have a high amount of salt or things like that and we forget to include atleast half of this concentration into the cryo solution.  This could easily crack the crystals depending on the concentration and the type of compounds in it.

Regards,
Mathews

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From: David Schuller

One more thing you could try: high pressure cryo-cooling. Se any of a number of paperas by Chae Un Kim; e.g.

http://www.ncbi.nlm.nih.gov/pubmed/17452791

Acta Crystallogr D Biol Crystallogr. 2007 May;63(Pt 5):653-9. Epub 2007 Apr 21.

--  ======================================================================= All Things Serve the Beam =======================================================================                                David J. Schuller                                modern man in a post-modern world                                MacCHESS, Cornell University                                schuller@cornell.edu 

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From: Jim Pflugrath


For some ideas on cryocrystallography, one can watch an online webinar on the subject:
http://www.rigaku.com/protein/webinar-001.html

Maybe some "unbiased" folks can comment? ;)

Jim



...
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From: Jens Kaiser

Hey Len,
 I had this problem, too. As you know, my favorite first try is always
fomblin (no need to mix anything). I had quite a bit success in stubborn
cases to inject about 4uL fomblin through the tape on top of the drop
and then looping crystals through the oil layer. You can wick the mother
liquor off and try them right away or continue manipulation under oil

Cheers,

Jens

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From: James Holton


I have always been a fan of oil, which has already been suggested.  Have you tried that?

Cross-linking has already been suggested, and these are some good protocols:
Lusty (1999) J. Appl. Crystallogr. 32, 106-112.
McWhirter, et al. (1999) PNAS USA 96, 8408-8413.

In the latter paper the crystals cracked immediately upon breaking the seal on the cover slip (limbo trays).  The cross linker was introduced with a Hamilton syringe via a pre-cut and grease-filled hole in the crystallization chamber.  That way there were no mechanical vibrations at all.  You do NOT need to add it directly to the drop.  Gluteraldehyde has sufficient vapor pressure to permeate slowly into it.  After a few days, the crystals were incredibly robust, and gave the best diffraction.  The only problem with gluteraldehyde is if you have primary amines in your buffer, such as tris.  If that is the case, you can usually substitute bis-tris, or just use a different kind of crosslinker.

Another trick I like if you have a "cryo" component in the mother liquor (protein counts) is to just let the drop dry up slowly.  You can keep "sampling" it with a small loop (removes ~1 nl) until you see it flash-cool clear.  Then, if the crystals survived, you can flash-cool them in the dried-down mother liquor.  This worked for me once with crystals that just didn't want to transfer into anything.

-James Holton
MAD Scientist