In the 1975 paper, they describe taking crystals to -100C, but it wasn't done in a
"flash" sort of way. They equilibrated the crystals with various solvent
combinations as the temperature was reduced.
Trying to recollect what was discussed by my lab mates nearly 40 years ago, I
think the fact that the crystals were mounted in capillaries caused
difficulties with plunging the crystals into liquid N2. We never tried it, but
the thought of what would happen to the glass, etc was enough to keep us from
doing the experiment.
I think the reason it took years before people started routine flash cooling of
crystals was because it was expensive and hard to do. You needed to buy a
crystal cooling device, and you had to invest the time and energy in developing
cryo-solutions. People were more excited about seeing the next interesting
structure, and room temperature experiments were good enough for that.
Ron
On Fri, 16 Nov 2012, Gerard Bricogne wrote:
Dear Quyen and Ron,
Thank you for bringing up this work. I can remember hearing Greg Petsko
give a seminar at the LMB in Cambridge around 1974, but I never read that
paper. The seminar was about cooling crystals at 4C, and also about work
done with Pierre Douzou to try and retain the high dielectric constant of
water (e.g. with DMSO) when cooling hydrated crystals to temperatures well
below the normal freezing point of water. This had to be done progressively,
with successive increases in the concentration of DMSO, and without ever
giving rise to a transition to a solid phase of the solvent; so it would
seem to have lacked the "flash" component of today's methods.
It may well be that their work went further into crystal cryo-cooling
and vitrification, and that this extension was described in the JMB paper
you quote but not yet in the version of his seminar that I heard. If so,
thank you for pointing out this reference - but then, why wasn't it taken up
earlier?
With best wishes,
Gerard.
--
On Fri, Nov 16, 2012 at 01:37:58PM -0500, Quyen Hoang wrote:
I was going to mention that too, but since I was a postdoc of Petsko my words
could have been viewed as biased.
Quyen
On Nov 16, 2012, at 1:26 PM, Ronald E Stenkamp <stenk...@u.washington.edu>
wrote:
I'm a little confused. Petsko and others were doing
low-temperature/freezing/vitrification crystal experiments in the 1970s, right?
(J. Mol. Biol., 96(3) 381, 1975). Is there a big difference between what they
were doing and what's done now.
Ron
On Fri, 16 Nov 2012, Gerard Bricogne wrote:
Dear all,
I think we are perhaps being a little bit insular, or blinkered, in
this discussion. The breakthrough we are talking about, and don't know how
to call, first occurred not in crystallography but in electron microscopy,
in the hands of Jacques Dubochet at EMBL Heidelberg in the early 1980s (see
for instance http://www.unil.ch/dee/page53292.html). It made possible the
direct imaging of molecules in "vitrified" or "vitreous" ice and to achieve
higher resolution than the previous technique of negative staining. In that
context it is obvious that the vitreous state refers to water, not to the
macromolecular species embedded in it: the risk of a potential oxymoron in
the crystallographic case arises from trying to choose a single adjective to
qualify a two-component sample in which those components behave differently
under sudden cooling.
I have always found that an expression like "flash-frozen" has a lot
going for it: it means that the sample was cooled very quickly, so it
describes a process rather than a final state. The fact that this final
state preserves the crystalline arrangement of the macromolecule(s), but
causes the solvent to go into a vitreous phase, is just part of what every
competent reviewer of a crystallographic paper should know, and that ought
to avoid the kind of arguments that started this thread.
With best wishes,
Gerard.
--
On Thu, Nov 15, 2012 at 11:35:46PM -0700, Javier Gonzalez wrote:
Hi Sebastiano,
I think the term "vitrified crystal" could be understood as a very nice
oxymoron (http://www.oxymoronlist.com/), but it is essentially
self-contradictory and not technically correct.
As Ethan said, "vitrify" means "turn into glass". Now, a glass state is a
disordered solid state by definition, then it can't be a crystal. A
vitrified crystal would be a crystal which has lost all three-dimensional
ordering, pretty much like the material one gets when using the wrong
"cryo-protectant".
What one usually does is to soak the crystal in a "cryo-protectant" and
then flash-freeze the resulting material, hoping that the crystal structure
will be preserved, while the rest remains disordered in a solid state
(vitrified), so that it won't produce a diffraction pattern by itself, and
will hold the crystal in a fixed position (very convenient for data
collection).
Moreover, I would say that clarifying a material is vitrified when
subjected to liquid N2 temperatures would be required only if you were
working with some liquid solvent which might remain in the liquid phase at
that temperature, instead of the usual solid disordered state, but this is
never the case with protein crystals.
So, I vote for "frozen crystal".-
Javier
PS: that comment by James Stroud "I forgot to mention that if any
dictionary is an authority on the very cold, it would be the Penguin
dictionary.", is hilarious, we need a "Like" button in the CCP4bb list!
--
Javier M. Gonzalez
Protein Crystallography Station
Bioscience Division
Los Alamos National Laboratory
TA-43, Building 1, Room 172-G
Mailstop M888
Phone: (505) 667-9376
On Thu, Nov 15, 2012 at 2:24 PM, Craig Bingman <cbing...@biochem.wisc.edu>wrote:
"cryopreserved"
It says that the crystals were transferred to cryogenic temperatures in an
attempt to increase their lifetime in the beam, and avoids all of the other
problems with all of the other language described.
I was really trying to stay out of this, because I understand what
everyone means with all of their other word choices.
On Nov 15, 2012, at 2:07 PM, James Stroud wrote:
Isn't "cryo-cooled" redundant?
James
On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote:
Perhaps it's an artisan organic locavore fruit cake.
Either way, your *crystal* is not vitrified. The solvent in your
crystal might be glassy but your protein better still hold crystalline
order (cf. ice) or you've wasted your time.
Ergo, "cryo-cooled" is the description to use.
Phil Jeffrey
Princeton
On 11/15/12 1:14 PM, Nukri Sanishvili wrote:
s: An alternative way to avoid the argument and discussion all together
is to use "cryo-cooled".
Tim: You go to a restaurant, spend all that time and money and order a
fruitcake?
Cheers,
N.
--
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