Hi Anamika, 1) What is the conc of PEG3350, PEG6000 in the crystallization drop? If high enough, you may not need additional cryoprotectant. Just try freezing intact crystals without additional cryo and collecting data.
2) Grow the crystals in presence of ethylene glycol or glycerol (i.e. add cryo in crystallization solution). You can also try to rescreen with some commercial crystallization kits that include cryoprotectant. 3) I presume you see the cracking during single step of cryoprotectant soaking. Have you tried serial soaks in increasing amounts of cryo, say from 1-16 or 18% EG, GOL in steps of few % and short soaks at each step? The gradual transition may be better in your case. 4) You can try to slow down the crystallization time (conc, temp), which may give you larger/thicker crystals (or even try seeding), which may lead to more durable crystals that survive the cryoprotection soak. 5) You should probably try some diffraction (maybe just 1-2 images) without any additional cryo and/or capillary mount in mother liquor to see what your resolution really is and see what is the impact from the cryoprotection step. Best, Debanu. -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Anamika Singh Sent: Thursday, April 10, 2014 11:00 AM To: [email protected] Subject: [ccp4bb] About crystallization diffraction problem Dear All, I want to get some help regarding crystallization for one of the protein I am working. This is a recombinant protein of molecular weight of 17.5 kDa. I am getting crystals shape like thin plates in 5 days, in different conditions like bis tris pH 6.5-8, HEPES pH 6-8 with .1M nacl , .01M DTT having PEG 3350, PEG 6000 as precipitant. But whenever we used to put crystal in cryoprotectant like 20 % ethylene glycol, glycerol, MPD it used to split like layers of some tree barks. And the crystal which were diffracting not getting diffracted above 3.0 Angstrom. Please help me out. Thanks in advance -- Anamika
