High-Pressure Freezing

Bal-Tec HPM 010 high-pressure freezer.

Assembly in the HPF specimen holder: quartz capillary containing the crystal (blue) in its mother liquor. The capillary is submerged in 1-hexadecene and sandwiched between two aluminium platelets.

Diffraction image of a Bovine enterovirus 2 crystal high-pressure frozen without any cryoprotectants (data collected at beamline I24 at Diamond Light Source). No ice rings were observed. The crystal diffracted down to ~2.5 Å resolution and possessed mosaic spreads of about 0.26°.

(in collaboration with the Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology in Hamburg)

Motivation
Macromolecular crystals typically contain between 30 and 90% of solvent (mainly water). Direct cooling of such crystals leads to the formation of crystalline ice which disrupts the crystal lattice. Successful cryocooling therefore requires the conversion of the water to amorphous ice (vitrification). This is normally achieved by exposing the crystals to penetrating cryoprotectants, e.g. glycerol or ethylene glycol prior to flash-cooling. Finding optimal cryoconditions can be very time- and crystal-consuming, especially for complex systems with large unit cells, such as membrane proteins or viruses. Even if adequate cryoconditions have been found, the crystal quality is often degraded upon conventional flash-cooling.
High-pressure freezing (HPF) allows sample vitrification without any cryoprotectants. This technique is well established in the field of biological electron microscopy for vitrification of cells and tissue and was recently optimized for macromolecular crystals by our group.

High-Pressure Freezing Procedure
Macromolecular crystals are either grown or soaked into quartz capillaries or cellulose microtubes and high-pressure frozen directly in their mother liquor. For HPF the samples are sandwiched between two aluminium platelets subjected to a pressure of 210 MPa while rapidly cooled to 77 K with ~7000 K/s using a Bal-Tec HPM 010 instrument. Sample mounting after HPF is carried out at cryogenic temperatures below 135 K. Our HPF protocol was successfully applied to several test systems, such as hen egg-white lysozyme and cubic porcine insulin [1], as well as the membrane protein photosystem II and Bovine enterovirus 2 [2], both being representatives for large unit cell systems.

References
1) Fast high-pressure freezing of protein crystals in their mother liquor,
A. Burkhardt et al., Acta Cryst. F68, 495-500 (2012).
2) Structure determination from a single high-pressure-frozen virus crystal,
A. Burkhardt et al., Acta Cryst. D69, 308-312 (2013).

For further information please contact:

Burkhardt
Anja Burkhardt
E-Mail: Anja Burkhardt
Phone: +49 (0)40 8998 1775