Have you tried to grow the crystals in the presence of small amounts of
cryoprotectants?
For example, use sucrose, glycerol, ethylene glycol, trehalose, glucose,
sorbitol, various salts in the crystal growth.
One can use concentrations of the additives in the 1% to 4% (w/v or v/v)
range. The hypothesis you are testing is this:
Does the additive in the tested concentration have any effect on crystal
growth?
If yes, don't use that additive. If no, go onto the next test: At what
higher concentration does the additive have any effect on crystal growth?
You can imagine where this leads. Perhaps once grown in a little bit of
cryoprotectant, then swishing through a higher concentration will not cause
cracking.
Also, do you suspect that the cryoprotectants that you have used bind to
your protein? Do they by chance mimic substrate, product, or effector?
And then there is always cross-linking with glutaraldehyde.
Good luck!
Jim
_____
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of
Fengxia Liu
Sent: Tuesday, September 01, 2009 5:25 AM
To: [email protected]
Subject: [ccp4bb] cracking crystal
Dear All,
Could you please help me solve this problem?
I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM
Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put
it in cryoprotectants, cracking happened. First i used artificial mother
liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all
crystals cracked after immersed, finally i tried 50% mineral oil + 50%
paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked
crystal can give me 3.2 angstrom diffraction, so no cracked crystal might
give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so
many cryoprotectants, so anybody has experience on this? any suggestion
would be greatly appreciated.
Thank you.
Fengxiale
