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freezing xtals -- a summary

LAWSON at BNLCL1.BNL.GOV LAWSON at BNLCL1.BNL.GOV
Mon Sep 27 14:32:13 EST 1993


As promised, here is a summary of the responses to my query regarding
freezing of protein crystals.

----------------------------------
The original query:

From:     IN%"LAWSON at BNLCL1.BNL.GOV" 19-AUG-1993 17:07:43.41
To:  IN%"xtal-log at net.bio.net"
Subj:     freezing protein crystals

Dear crystallography netters,

We at Brookhaven have finally joined the modern world with the purchase
of an Oxford Cryostream system to immortalize our protein crystals in
the X-ray beam.  We ourselves have little experience with freezing crystals,
but know that there are several labs who are gradually gaining expertise.
We would appreciate hearing about any tips or positive/negative experiences
with freezing crystals, including (1) how to mount crystals, (2) freezing
in liquid propane or liquid nitrogen first vs. putting the crystal directly
in the cooling stream (3) types of materials used to coat the crystal
and (4) use of anti-freeze additives.  I suspect there may be enough
interest in this topic to post responses directly to this news group, but
private responses are also most welcome.  I will eventually post a
summary of all responses.

Thanks!

Cathy Lawson
Biology Department,  Brookhaven Nat'l Lab, Upton NY   11973
lawson at bnlcl1.bnl.gov
(516) 282 - 7667
------------------------------------------

Unfortunately, the most valuable responses for me will be less
helpful to interested netters, as these consisted of two people who
graciously offered to tell and/or show me their methods. It is 
somewhat embarrassing that one of these people is someone who also
works at Brookhaven, on the Argonne protein crystallography
beamline, so I should have known to contact him before my query 
but didn't think of it!  Anyway, I still plan to take you up on your 
invitation, Steve (it's been a hectic month):

-------------------------------------------
From:     IN%"GINELL at BNLSBC.NSLS.BNL.GOV"  "STEVE 282-3018" 19-AUG-1993
Cathy....hi
1) At Argonne National Labs Structural Biology NSLS beamline X8C we have 
     been using the LN2 cooled data collection methods (-170C) 
     on a routine basis for nearly one year (Oct 1992). These methods have 
     proved invaluable in collecting Synchrotron X-ray data sets on a 
     single crystal 
2) the methods of mounting xtls have included glass fibers and plates, nylon,
     glass, silk, metal, and other synthetic materials formed into loops 
     and cages
3) cryo solvents/protectents used have been of alcohols, glycerol, oils, 
     greases, and other non-salt solutions 
4) methods of freezing have includes direct freezing in the cold stream,
     quenching in liquid propane or liquid nitrogen
5) mounting methods have included glass fibers, and metal pins,
     mounted on both magnet and nonmagnetic bases 

I invite you to stop by X8C sometime after 6Sept93 to see how
things are set up.

Stephan Ginell

(516)282-3018
---------------------------------------
The other person I will not name as he has not wished to be part of
this public forum.  I would, like however, to mention one of the most
valuable things I have learned from him so far:  He suggests that
before testing a valuable crystal, one can test-freeze droplets of
a crystal mother liquor.  If the droplet is still clear after freezing,
probably no ice has formed and one can then proceed with the crystal.
If the droplet clouds up, one can test variant mother-liquors with
cryosolvents until one finds a non-icing condition.  This, he warns,
is really only a rough guide, but it certainly seems like a simple
and useful experiment to try before sticking your precious
crystal in front of a cryostream.

To encourage anyone else to STILL contribute
tips on crystal freezing, here are some readers of this bionet 
newsgroup who have open ears:
---------------------------------------
From:     IN%"DRESS at biosci.arizona.edu" 19-AUG-1993 18:16:09.69
     We have just started trying to get our frozen crystal system set-up
also, so we would appreciate it if you could pass any tips to us.         
Virginia Dress
---------------------------------------
From:     IN%"bburkhar at magnus.acs.ohio-state.edu"  "Brian M Burkhart" 
Could you please forward any information you get in this matter. I am quite
interested in this sort of experimentation.
many thanks 
Brian Burkhart (send to brian%biot at mps.ohio-state.edu)
---------------------------------------


There ARE a few tips I received that are worth passing on:

---------------------------------------
From:     IN%"xth at mace.cc.purdue.edu" 20-AUG-1993 20:03:03.83
I am a Research Associate in the Crystallography group at Amgen.  We
have successfully done this for three protein crystals.  In each we used
anti-freezing additives such as glycerol and PEG 400. In one case the 
crystal was grown in 30% PEG 400 so we were able to freeze it directly.
One of them was grown in 30% PEG 4K.  We slowly removed the solution
from around the crystal and added the PEG 400.  In the third we used
30% Glycerol.  In each case the crystal did not crack (much) and we 
were able to collect data using liquid nitrogen.  It substantially
improved the diffraction,  but increased the mosaicity.   Needless to
say we went through many crystals to get this to work.  I think it's
best to grow the crystal directly in PEG 400.
     
The crystals were mounted in a loop of hair.
  
I hope this helps.

Elizabeth Singer
X-Ray Crystallography Lab
Amgen
---------------------------------------
From:     IN%"GREGERS at kemi.aau.dk" 26-AUG-1993 11:23:20.46
To:  IN%"lawson at BNLCL1.BNL.GOV"
With regard to low temperature, we have in our lab  very promising 
results. We have sucesfully frozen and collected data from several
types of crystals.Our general results are:

1) Mounting. We have almost always
used the loop method. It is very easy
to perform.So far we have used metal wires, but we are currently
attempting with surgery thread.
2) Freezing meth. So far we have only used freezing in the cryo
stream, but we will try to use freezing in propane in the future.
It is our experience, that 2/3 of the experiments fail due to 
bad freezing, which is clearly seen as a high background/high mos.
3) Coating material. If this is the oil method, we have little
succes. One reason could be that many of the crystals were 
quite fragile.
4) Anti freeze additive.30 % Glycerol seems to be a good choice.
Goes along with even 40% AmS. The solubility generally increase when
adding glycerol. The problem can be solved by using short
soaking times. In one case, we transferred xtals from mother liquer
directly to the soaksolution and soaked them for 30s with good
result.

Gregers 




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