Have you tried 3.5M Ammonium Sulfate? This is a very good
cryoprotectant and I am often surprised that many people don't seem to
know this. Many salts (with low thermal coefficient of solubility) are
good cryos at near saturation. You don't want to be at saturation with
NH4SO4 because a small amount of evaporation will give you a shower of
hexagonal rod salt crystals, and this presents obvious problems if your
protein crystals happen to be hexagonal rods.
The other problem with saturated NH4SO4 is that it absorbs about 3.2x
more x-rays than water (assuming you add 0.77g to 1 mL H2O to get 1.42
ml of saturated solution). This means that your crystals will probably
burn up 3.2x faster than if they were in a water/organic cryo. However,
I have personally noticed that water/organic cryos will expand visibly
when dosed with x-rays whereas near-saturated NH4SO4 cryos do not. It
is possible that this volume change upon irradiation is part of the
radiation damage process (distorting the crystal lattice), so there may
be some compensating advantage to using salty cryos. However, I don't
know of anyone who has studied this systematically (including me).
It is interesting that you get isotropic diffraction at RT in
capillaries. Have you tried freezing in capillaries? You can get away
with less cryo than usual doing this (the glass acts like a layer of
oil). 2.5 M NH4SO4 might even be enough. The only trick is mounting up
a short sealed capillary on a cryo pin.
-James Holton
MAD Scientist
Ji lee wrote:
Dear,
I have a crystal diffracted anisotrophically. I tested with a few
different cryo conditions like oil, glycerol in different
concentration to get a better data but these conditions didn't help
any.
Using capillary method improved the diffraction (isotrophic) but the
crystal couldn't survive during the data collection.
Is there any methods or cryo conditions I can use to improve my
diffraction data?
This crystal grew in 2.5M Ammonium Sulfate.
Thank you so much.
Ji
