Keith,

There could be as many number of stories of cryoprotection to as many number of
structures deposited in the pdb.

PEG3350 seems to be little too large for freezing, particularly in presence of so much
of salt. I have collected data up to 1.0 Ang with PEG2000 freezing.

If you have not tried:
-try oils

-try from low concentrations of cryoprotectants to the required concentration in steps
I have seen people do this process over several hours to several days. 

-Since your crystals seems to already have some glycerol, it may be worth to try that in
steps of 2-5% up to 20-25%. Larger the crystal longer should be the incubation in each step.

-From my experience, on freezing, small crystals do better in terms of mosaicity. If you
plan to collect data on a synchrotron, choose small crystals.

Good luck

Anthony


On Sat, 14 Feb 2009 21:15:11 -0500, Keith Romano wrote
> Hi all,
> 
> I have protein crystals in complex with substrate grown in 20-30% (w/ 
> v) PEG 3350, 4% (w/v) ammonium sulfate, and 0.1M sodium MES buffer at  
> pH 6.5.  I purify and concentrate my protein in a high salt buffer  
> (0.5M NaCl, 0.1M sodium MES at pH 6.5, 10% glycerol, 2mM DTT).
> 
> I grow my crystals with vapor diffusion in 24-well format by hanging  
> a drop of equal volume protein and precipitant solution over the  
> reservoir of precipitant solution.  Interestingly, when I do the  
> math, the initial osmolarity of my drop is greater than that of the  
> reservoir (due to the high NaCl in the protein solution).  As far as  
> I know, this runs against the principles of the vapor diffusion  
> method, as vapor will leave the reservoir and enter the drop...
> 
> Nevertheless, these conditions yield giant, rod-like crystals over  
> 1mm long.  However, they don't react well to direct flash freezing-  
> the spots tend to smear and indexed refinement leads to high  
> mosaicity.  I have tried many cryoprotectant solutions by making up  
> the given precipitant solution with 15-25% glycerol or ethylene  
> glycol, including a range from 0mM to 350mM NaCl.  In general,  
> dipping the crystals in cryoprotectant improves the diffraction and  
> lessens the "spot smearing".  However, the diffraction usually  
> becomes highly twinned and hard to index.  After transfer into the  
> cryoprotectant, the crysatls appear to crack and often break apart,  
> as observed under the microscope.
> 
> It seems like my crystals are very sensitive to the osmotic/ionic  
> change when transferred to the cryoprotectant.  I have been unable to  
> find a "stable" cryoprotectant, and I am wondering if anyone has had  
> similar experience with high-weight PEGs and could suggest some  
> cryoprotectants to try out.
> 
> Any input would be greatly appreciated!
> 
> Keith
> 
> Department of Biochemistry & Molecular Pharmacology
> 970L Lazare Research Building
> University of Massachusetts Medical School
> 364 Plantation Street
> Worcester, MA 01605


-------------------------------------------------
Anthony Addlagatta, Ph.D.
Ramanujan Fellow and Senior Scientist
Center for Chemical Biology
Indian Institute of Chemical Technology [IICT]
Tarnaka, Hyderabad- 500007, INDIA
Tel:+91-40-27191583
Url: http://www.iictindia.org/zacb/Dr.%20Anthony.aspx