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 <[log in to unmask]> 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 <[log in to unmask]>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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