Dear Fengxiale,
I would suggest two thing you can try:
1) shock-freeze the crystal: pick it up from the crystallisation plate
with the mounting loop and only briefly (<=1s) dip it into the
cryo-condition, then freeze it in a bath of liquid nitrogen.
I would try both glycerol and PEG400.
- try to find the minimum required amount of glycerol and PEG400 by
testing various conditions withouth the crystal: freeze the cryo-solution
in a large loop and take a picture. Go high enough with the cryoprotectant
to avoid any ice-rings at all.
2) transfer the crystals _really_ slowly!
Make solutions in steps of 5% cryo-protectant in the mother liquor and
transfer the crystal with a pipette. If the crystal is too big to go
through the pipette tip, cut off a small piece with a scalpel.
After each step observe the crystal, check whether you observe any
cracks. Wait for 5-10min before going to the next concentration.
This way you can check various cryo-protectants like PEG400, MPD,
glycerol, sugars (fructose, sucrose)...
Since you don't have to many crystal, you can use the cracked parts for
this experiment, so you only have to sacrifice one or two crystals.
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Tue, 1 Sep 2009, Fengxia Liu wrote:
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
