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
