Procedure of HPC method:
- Pick up a crystal with a cryoloop, coated with oil.
- Loading it into a high pressure tube, hold with a magnet.
- Pressurize the sample with Helium gas (200-400 MPa).
- Release the magnet, let the sample fall down to the bottom of the high pressure tube (200-400MPa).
- Unload the sample in liquid nitrogen (0.1MPa).
- Mount the cryoloop to a base. Then proceed with collection of diffraction data, using the same methods as for non-pressurized crystals.
Cryocooling under high pressure was first carried out during the initial development of low temperature protein crystallography but subsequently neglected due to the development of cryoprotectants. We have now returned to the technique and, over the last several years, have developed HPC into a useful alternative to standard cryocooling. For this technique, crystals (or other samples) are pressurized with helium gas, typically to 200-400 Mpa (2-4 kbar), and cooled to liquid nitrogen temperature under pressure. The pressure is then released and no special handling is required thereafter. As long as the temperature remains below 100 K, the effects of pressure cooling are “frozen in”. By using HPC, damage due to cryocooling may be significantly reduced, and penetrating cryoprotectants are often not required. Data obtained from pressure-cryocooled crystals can be of very high quality, and high resolution structures have been obtained using HPC. The protective effect of pressure during cryocooling appears to result from the conversion of the water in macromolecular crystals from low-density amorphous (LDA) to high-density amorphous (HDA) ice.
There are three pressure cryocooling systems with different maximum pressure available for HPC at MacCHESS:
HPC system @200MPa: this system has a maximum pressure of 200MPa, for routine work, such as reducing cryocooling-induced damage.
HPC system @400MPa: this system has a maximum pressure of 400MPa, for work requiring higher pressure. This is sometimes the case when the goal is to reduce disorder present in the original crystals.
A detailed protocol for HPC has been developed; this process is carried out by MacCHESS staff.
Lower-pressure cryocooling system: pressure usually used is 5-20 MPa, for gas trapping:
a) Gas-loading and Cryo-cooling apparatus and b) Schematic overall view.
