Actually, there is a particular kind of "freezing" than can be a good
thing: cubic ice. The specific volume of cubic ice is about 2% higher
than that of amorphous solid water (or hyperquenched glassy water). In
cases where the "preferred" specific volume of the protein lattice is a
little bigger than the "preferred" specific volume of the stuff in the
solvent channels upon vitrification, a small amount of cubic ice
"character" can actually make your crystal diffract better.
Basically, a mis-match in the "preferred" volume of the protein and
solvent creates stress, and stress makes high-angle spots go away (see
Juers et al. 2001, 2004). I think this is one of the most
under-appreciated things about screening for cryos, and seems to be the
main cause of that mythical "one crystal in a thousand that diffracted
well" problem. In my experience, this means you are being very
consistent in your cryo-cooling but you made one mistake. Final density
upon flash-cooling is VERY sensitive to the cooling rate, and little
things like the thickness of the layer of cold N2 gas above your liquid
N2 can change the cooling rate by a factor of 100 or so (Warkentin et
al. 2006).
You can tell if you've got some cubic ice character by looking at the
background of your diffraction pattern. Hexagonal ice has three rings
at 3.45, 3.68, and 3.91 A, whereas cubic ice only has the 3.68 A ring.
Moreover, noone has ever grown a macroscopic crystal of cubic ice, and
the smaller the crystallites get, the broader the 3.68 A ring becomes.
Eventually, it can be hard to tell the difference between
nano-crystalline cubic ice and the diffuse background expected for
"amorphous" solvent. You have to look carefully at the width of the
"water ring", because the first major diffuse ring from amorphous solid
water is also at 3.68 A. In fact, all the major diffraction peaks of
cubic ice are precisely centered on the diffuse rings of amorphous solid
water. This is perhaps not a coincidence, but it does mean that there is
a continuum of states between a sharp "ice ring" and an amorphous "water
ring". Your best diffraction may well be somewhere in the middle.
-James Holton
MAD Scientist
On 11/15/2012 10:12 AM, A Leslie wrote:
Dear Sebastiano,
This is not entirely straight-forward.
The Oxford English dictionary gives the first definition of "freeze"
relevant to this discussion as:
"Of (a body of) water: be converted into or become covered with ice
through loss of heat"
This is certainly not what we want to do to our crystals.
However, another definition in OED is:
"Cause (a liquid) to solidify by removal of heat", suggesting that
this does not necessarily mean the formation of crystals.
The Larousse Dictionary of Science and Technology (1995) has the
following definition:
"Freeze-drying (Biol.) A method of fixing tissues sufficiently rapidly
as to inhibit the formation of ice-crystals."
The Dictionary of Microbiology and Molecular Biology (3rd Ed) in the
entry on "Freezing" has the sentence:
"Rapid freezing tends to prevent the ice crystal formation by
encouraging vitrification".
Both of these erstwhile volumes therefore suggest that freezing does
not necessarily imply the formation of crystals. However, the term is
ambiguous, while vitrification is not.
Personally I use "cryocooled" instead.
Best wishes,
Andrew
On 15 Nov 2012, at 17:13, Sebastiano Pasqualato wrote:
Hi folks,
I have recently received a comment on a paper, in which referee #1
(excellent referee, btw!) commented like this:
"crystals were vitrified rather than frozen."
These were crystals grew in ca. 2.5 M sodium malonate, directly dip
in liquid nitrogen prior to data collection at 100 K.
We stated in the methods section that crystals were "frozen in liquid
nitrogen", as I always did.
After a little googling it looks like I've always been wrong, and
what we are always doing is doing is actually vitrifying the crystals.
Should I always use this statement, from now on, or are
there english/physics subtleties that I'm not grasping?
Thanks a lot,
ciao,
s
--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy
tel +39 02 9437 5167
fax +39 02 9437 5990
please note the change in email address!
[email protected] <mailto:[email protected]>
