This is generally the result of strain upon cooling. I. E. the outside
of the crystal is shrinking and the inside is expanding. The trick is
to match the density of the stuff in the solvent channels upon cooling
to the preferred final density of the lattice (Juers and Matthews,
(2004) Acta D60, 412). The problem is that there is no way to know what
your lattice's preferred density is, so you have to screen. This is
also harder with bigger crystals (hundreds of microns across) because
the inside of such crystals cannot flash-cool at the same rate as the
outside, due to the thermal conductivity of the crystal itself.
The fastest cooling rates have been achieved by blowing off the gas
layer on liquid nitrogen during plunge cooling (Warkentin et al. (2006).
J. Appl. Crystallogr. 39, 805). This is very easy to do!
It is a pity you did not mention which salt, as many salts are
"cryosalts": malonate, formate, ammonium sulfate (at >3.5 M), lithium
sulfate, and perhaps others. Anything with a flat solubility vs
temperature is a good candidate. Mixing of different cryos can often
have a superior protective effect to single-component cryos of the same
total concentration (the "confusion principle"), so there are a lot of
combinations to try.
Cooling, as well as soaking in stuff can often lead to osmotic shock
(Garman (1999) Acta D55, 1641). You can avoid this by calculating the
osmotic pressure of your cryo, or perhaps by equilibrating a small
droplet of it via vapor diffusion with the reservoir/mother liquor for a
few days. Another trick is to slowly concentrate the mother liquor by
leaving the drop open to air and allowing it to slowly dry down,
checking the mother liquor for "freezing clear" every few minutes. You
do not need an X-ray setup to check for "freezing clear", you can do
this by putting the dewar under an ordinary dissecting scope with
overhead lighting, focusing on the surface of the liquid nitrogen, and
bringing the flash-cooled loop into view just above the liquid surface,
where it is still very cold.
You could also try cooling the crystal slowly, which can be done using
fomblin oil (Warkentin & Thorne (2009). J. Appl. Crystallogr. 42, 944),
or by a schedule of substituting methanol for water to keep the
dielectric constant at 80 (Petsko (1975). J. Mol. Biol. 96, 381).
The fact that you get no diffraction in paratone-N implies that your
solvent content is high. Do you see ice rings? If so, this is because
ice nucleated inside the solvent channels (which can happen if the
solvent content is > 50%), or you missed a tiny volume of mother liquor
that was still clinging to the crystal surface when you cooled it, and
this will surely nucleate ice. The combination of oil and a small
amount of penetrating cryo can often be helpful in this case. Again,
this is combining two different cryos.
The only way you can be sure the oil (or any other cryo) is
fundamentally messing up the crystal is to shoot it in the oil at room
temp. These kinds of controls are essential for figuring out what your
crystal "likes" and does not "like". Flash cooling is generally not a
very reproducible process, especially if you are using oil and are not
careful about removing all the surface liquid. The level of nitrogen in
the dewar is also a major source of variability in cooling rate, and
cooling rate is a major determinant of the final density of the cooled
mother liquor.
The unreliable nature of flash cooling is perhaps why people continue to
"gamble" with it, hoping for a miracle event with an easy technique, but
there are plenty of alternative techniques that have been shown to work
better, albeit with more expensive apparatus. Cooling at high pressure
is a shining example of this (Thomanek et al. (1973) Acta A29, 263).
There are commercial high-pressure freezers (such as the Leica
EM-PACT2), and they do have a very successful high-pressure system at
Cornell (Kim et al. (2005) Acta D61, 881). Perhaps you could contact
the authors with your "test case"?
-James Holton
MAD Scientist
Natalie Zhao wrote:
> -----Original Message-----
> From: [log in to unmask] [mailto:[log in to unmask]] On Behalf Of Rafael Couņago
> Sent: 14 December 2009 20:22
> To: [log in to unmask]
> Subject: [ccp4]: TDS upon flashcooling
>
> Dear all,
>
> I got these beautiful looking crystals that grow in high salt (1.8M) and
> diffract under 2.0A at room temp. My attempts so far to cryo protect
> them have resulted in a loss of resolution (2.5A tops) and increased
> anisotropy.
>
> I have tried some of the usual suspects; no cryo, ethylene glycol,
> glycerol (even 5% makes my crystal crack), sucrose, glucose, paratone-n
> (no diffraction at all). I have tried both dipping the crystal straight
> into liquid nitrogen and flash cooling it in the cryostream.
>
> An interesting observation is that the diffraction pattern following
> freezing has a substantial amount of thermal diffuse scattering (but no
> ice rings). If I remove the crystal from the cryostream and re-anneal
> it at room temp (in air or in mother liquor or mother liquor + cryo)
> most of the TDS goes away, but the max resolution is still around 2.5A
> and the higher anisotropy is still there. Extending re-annealing times
> lead to cracking of the crystal.
>
> My two questions would be:
>
> - any thoughts on cryo solutions?
> - does the result from the re-annealing experiment ring any bells?
> Would this be an indication that I need the cooling to be faster or slower?
>
> Cheers,
>
> Rafael.
>
>
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