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[ccp4bb]: Cryo/Oil Summary
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Thanks to all for their contributions! It is clear from the
responses that oil is no panacea, but it seems to work very well
in many cases. We've had good luck so far, but organic solvents
in the drop may pose problems. We do see diffuse scattering due to Si,
but not enough to be concerned. Some suggest drying the oil as an
aid in removing the water layer on the surface of the crystal. We suspect
technique is very important here, and oil composition less important.
We tried a silcone-based diffusion pump oil from Dow (750).
It is thermally stable and claims to be radiation and oxidation resistant.
There is an older small-molecule reference I have not been able to locate, so perhaps
someone can forward it. Anyhow, here are the papers we've found so far (Thanks Ulrich Englich).
o S. Parkin and H. Hope, J. Appl. Cryst. (1998) pp945-953
(section 2.1 of this paper recommends Paratone-N, posibly saturated
with water. Recommends against Si or F -containing oils due to
higher scattering power. Half the xtals they have tried survive oil
treatment. Main problems are mechanical strength, loss of water by xtal
resulting in cracks???, or difficulty removing water layer. They are
advocates of quick-dunk cryoprotection when oil does not work.
o H. Hope, Annu. Rev. Biophys. Chem. 1990 19:107-126
more details of oil/cryo handling (covering hanging drop with oil
and dragging xtal through oil-water phase, wicking etc.)
o Riboldi-Tunnicliffe and Hilgenfeld J. Appl. Cryst. (1999). 32, 1003-1005
Harry Powell <email@example.com>
This is an excellent method and has been used for many years by small
molecule crystallographers for freezing extremely air-sensitive crystals.
I've used it successfully with macromolecular crystals too.
I've used a perfluoropolyether oil for this (used to be Riedel-de-Hahn
RS3000, but this hasn't been manufactured for many years. I haven't needed
any since '95 so haevn't looked into it seriously, but new sources have
been discussed on this BB in the last year or so).
For the small molecule case, it works by providing a physical barrier -
the amount of oxygen that can diffuse through the oil os actually quite
small. Also, something I didn't mention before - most air-sensitive
compounds are actually sensitive towards hydrolysis, so it isn't the
oxygen that reacts directly with them. Water, of course, is not terribly
soluble in perfluoropolyethers. However, nothing which isn't pfpe is
soluble in pfpe oils.
For macromolecules, it stops evaporation of water from the crystal, giving
you time to cool to create a vitreous phase. But the migration of oxygen
through the oil is also limited, so that helps too.
we have used MO (mineral oil) only occasionally and with indifferent results.
that is, sometimes we get useful freezing but never better
diffraction. we purchased a panjelly kit and tried their
[ the pan jelly kit consists of 10 small containers of
pan jelly, a gooey mixture of some sort ,not published;
a small amount of very thin dried mineral oil;
acouple of depression slides and an alleged glass spatula]
the main selling point ,for me at least, was the supposed
improved reliability of annealing protein crystals (something
we have had no luck with , whatsover). following their
suggestions we got poor results. nothing (including
lysozyme) diffracted any better than we had obtained by
conventional means and in no way did we find any help
annealing crystals. add to this that the stuff does no
not perform well in the cold room we let it languish on the
shelf for some months.
recently I had an annoying problem with a crystal that had
frozen well for about 2 years worth of xray experiments but
then for some reason refused to freeze usefully . I turned
to MO but that did not work very well either, the mother liquor
sticking to the crystal persistently. now, the pan jelly
is good at getting off the surface aqueus film but is
difficult to get rid of itself. also , these crystals
had to be manipulated in a cold room (panjelly becomes too
thick to manipulate a crystal into at all)
so, I tried taking a bit of somewhat dry pan jelly and
mixed it with a bit of MO to get a slightly pink soupy
mixture. I imersed my crystal in this, then into MO
to get rid of the pink soup leaving a very clean crystal
that froze well directly out of the crystalization droplet
as well as had the crystals under the old method. I have
done this now several times and it works well for this
system. on the other hand it has been tried on at
least 3 other crystals without any luck.
the nice thing is that crystals can be froze an lower
concentrations of cryo-protectant (in my case 30% as
opposed to 40-50% in the old technique)
I have been admonished to practice more with lysozyme
by the "parents" of pan jelly but enough is enough
I could not really get better results using hteir
methods. both their low viscosity MO and commercial
light mineral oil function about the same, I find
the slightly thicker light MO easier to retrieve
a crystal from and so tend to use it.
Dmitriy Alexeev <firstname.lastname@example.org>
I tried 3 different oils and their mixtures - all successful so far and now I always
use it by default. The first oil was the machine oil from the wokshop, the latest -
Paratone N. No special preparations were required.
Yours Dmitriy Alexeev.
Our laboratory has used oil, in place of a cryprotectant, for cubic lipid
phase bacteriorhodopsin crystals successfully...
We've tried oil once so far, on crystals of a rather
large protein-DNA complex grown from Ammonium sulphate. At room T, they
diffracted to 13A, and frozen in propane, 13A, but the ones we tried in oil
didn't yield a single spot (at a synchrotron). (And we did have help from
someone who swears by oil).
Now granted, these crystals seem to be useless no matter what we do, but
oil-freezing certainly didn't improve things!
Salam Al-Karadaghi <Salam.Al-Karadaghi@mbfys.lu.se>
Hopefully it is not a membrane protein you need to freeze, oil drop would
not work in this case for obvious reasons.
Alan Tunnicliffe <email@example.com>
Here is the reference for our paper using oil as a cryoprotectant
Riboldi-Tunnicliffe and Hilgenfeld
J. Appl. Cryst. (1999). 32, 1003-1005
"Zac A. Wood" <firstname.lastname@example.org>
The oil method has worked very well with four different crystals in my
hands, and it is now the first thing I try. It decreased mosaicity with
regard to other cryos in one case, and proved essential in freezing one
extremely fragile crystal without damaging it. The other advantage I find
is that you do not need a artificial mother liquor. I have also had one
crystal that it did not work with, so it is not always a sure thing. I
have a feeling that in that latter case it may have had something to due
with high solvent content. Briefly the technique I employ is as follows:
(for hanging drops)
1) cover the drop on the coverslip with a small amount of oil (20-40 ul).
When I first read of this technique, I was eager to try it a a troublesome
crystal and actually used fresh vacuum pump oil. it worked like a charm,
and I have used it since with no trouble.
2) with a loop, fish the crystal out. I like to use a loop smaller than
the crystal (spoon it). I get less of the mother liquor sticking
to the crystal/loop that way. I also find that it is not to difficult to
ge rid of any risidual mother liquor by passing the crystal back and forth
through the mother liquor/oil interface. I had trouble with this and
loops big enough to hold the entire crystal. The oil "glues" the crystal
to the loop.
3) plunge in liguid N2 or freeze in a stream. I usually plunge myself.
Jim Pflugrath <email@example.com>
Oils are great. We use perfluoropolyether,
paratone-N, and 75:25 or 50:50 paratone-N:mineral oil. At least in one
case where 100% paratone-N cracks the crystals, the 75:25 mixture worked.
adam staines <firstname.lastname@example.org>
I frequently use oils when using high salt precipitants as the phase
difference traps the salt in the crystal and stops diffusion between the
cryo protectant and the crystal.
I have found it usually works for most high salt crystals and some PEG
grown crystals as well.
The problem is the oils diffract and give diffraction rings.
I have always found parrafin oil (Hampton) works fine. It gives rings at ~4
and 2.3 A so a normal data set has only two rings. The rings are usually
quite small so I dont loose much data.
If it wasn't for the rings id use oil as first choise as it usually works
first time and therefore saves time fiddeling with cryoconditions.
recently I got three to work from: 4M NaFormate, 2.5M A.S. and 24%PEG grown