High Pressure Cooling

High Pressure cooling

A facility for cryocooling crystals under pressure is now available at MacCHESS. This technique, developed in the Gruner lab, was reported in (Kim et al., Acta Cryst. D61, 881 (2005)). It involves mounting a crystal on a special pin, pressurizing it, cooling to liquid nitrogen temperature, and then releasing the pressure while keeping the crystal cold. The method can allow successful cryocooling using little or no penetrating cryoprotectant, and can produce cryocooled crystals of better quality than the usual cryocooling method. In addition, pressure-cryocooling can act to stabilize a single conformation of a bound ligand, hence making it visible in an electron density map (Albright et al., Cell 126, 1147 (2006)). It is also possible to apply the method to samples in capillaries, both solutions and crystals mounted, or grown, in capillaries.

An apparatus for cryocooling samples under pressure has been installed at CHESS. It is designed to enclose all high pressure components in a steel safety container with 1/2 inch thick walls. Weight: ~3000 lb. Users can provide unfrozen crystals and request staff to pressure-cool them. Diffraction from the first user crystals processed at CHESS improved in resolution from 3.2 to 2.8 A, and images showed better spot shapes.

Steps in standard pressure-cooling:

• Mount sample (usually a crystal in oil, but can be anything that fits in the pressure tubing) on a special pin (1) with a piece of steel piano wire attached to the base.
• Slide pin with mounted sample into pressure tubing (2), where an external magnet holds it near the top.
• Place up to 3 tubes in a bath partially filled with liquid nitrogen (3).
• Place the bath in a safety enclosure (4).
• Connect tubes to a manifold (5) and pressurize system with helium, usually to about 200 MPa (2 kbar).
• Remove magnets, letting the pins fall to the bottom of the tubes, (at 77 K).
• Release pressure from the system.
• Disconnect tubes at top and bottom, keeping pins with samples under liquid nitrogen (6).
• Transfer pins to standard bases; then handle like any other cryocooled samples.