Re: [ccp4bb]: cryo-protect crystals grown in 35% dioxane

[Lisa Edberg <ledberg@uab.edu>]

***  For details on how to be removed from this list visit the  ***
***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***

Hong Zhang wrote:

> We just got crystals from 35% dioxane. Does anybody knows how to
> cryo-protect those crystals?
> The crystals deteriate after 1 week, so we have to save them fast.

Of course it depends on the protein and the crystal.

There is the possibility that these crystals may be able to be flash
cooled as is (remove the solvent from around the crystal), or, perhaps
after being dipped in paratone (the magic oil). If 35% dioxane freezes
clear (I doubt it) you're golden. Just go with that.

However, life is rarely that easy. You probably need to add a
cryoprotectant. Some have had luck with adding solid sorbitol to the
mother liquor. If you add a polyol such as glycerol, ethylene glycol,
mpd or peg 400, you may need to decrease the water in your mother liquor
so that the proportion of precipitant, dioxane, is the same or higher
than the conditions used to grow the crystals. For example, make up a
new buffer that is 37-40% dioxane, 10% glycerol, 50-53% water, rather
than mixing 10% glycerol and 90% well buffer. Many proteins become more
soluble in the presence of glycerol or ethylene glycol.

Test the conditions with an empty loop to find the minimal amount of
cryoprotection needed for your buffer. Once you find a condition that is
clear when cooled, you should go with that. Use the smallest possible
loop that will comfortably contain your crystal. The more solvent that
surrounds your crystal, the more solvent scatter, the more diffuse your
data.

I have had a crystals that would not survive even the gentlest of
transfers to cyoprotectant (adding 10% v/v of cryoprotectant buffer
slowly to the mother liquor, mixing, waiting, withdraw 10%, add 10%,
etc...). however, when I grew the crystals in the presence of a small
amount of glycerol- only 1%, I was able to transfer the crystals
directly to the cryoprotectant buffer containing 15% glycerol with no
ill effects.

Be prepared to use up a lot of crystals in these tests. Hopefully your
crystallization conditions are sufficiently refined so that you get
consistent results and don't waste protein on clear drops and needle
showers. The closer your condition is to an optimum, the better your
chances will be for getting reproducible results in every drop.

Good luck,

Lisa

PS Don't forget to collect complete, redundant, accurate data, and try
to collect as much low resolution data as you can.
--
Lisa Edberg (205)934-1611 FAX:(205)934-0480
UAB Center for Biophysical Sciences/Engineering
http://www.cmc.uab.edu/misc/edberg/index.html
"It's not always that Nature is being nasty to us.
Sometimes also we are stupid." -Thomas Schneider