High Pressure Cryocooling for Capillary Sample Cryoprotection and Diffraction Phasing at Long Wavelengths

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Chae Un Kim^1,2, Quan Hao², and Sol M. Gruner^1,3,4
1Field of Biophysics
²MacCHESS, Cornell University
³Cornell High Energy Synchrotron Source (CHESS),
  Cornell University
⁴Physics Department, Cornell University

Abstract:
Crystal cryocooling is usually employed to reduce radiation damage during x-ray crystallography. Recently a high-pressure cryocooling method has been developed which often results in better crystal diffraction than conventional cryocooling methods, even with the limited use of penetrative cryoprotectants. This method has been successfully extended to crystal diffraction phasing at a long wavelength (1.7 Å) and two recent results are presented here. First, xenon gas was incorporated in the high pressure cryocooling method and de novo Xe - SAD phasing was carried out on porcine pancreas elastase (PPE, 240 residues, 26 kDa). A single 0.70 occupied xenon site per PPE gave an expected Bijvoet amplitude ratio (<|ΔF|>/<F>) of 2.8 %. The auto-model-building with the experimental phases was very straightforward: 97 % of total residues (232 out of 240) could be found and docked in the electron density at 1.8 Å resolution. Secondly, native sulfur SAD phasing was performed on thaumatin crystal diffraction. Surprisingly, successful cryoprotection of the crystal was achieved in a thick-walled (300 µm) capillary without additional cryoprotectants other than the native mother liquor. All 17 sulfur atoms in thaumatin could be distinguished in the anomalous substructure and S - SAD phasing was successful at 1.9 Å resolution. These developments may be useful for the structural determination of proteins without the need for seleno-methionine incorporation and for high-throughput crystallography.

abstract (pdf)