Successful protein crystallography typically requires that crystals be cryocooled in order to reduce radiation damage at the data collection stage. So far, protein crystals have most commonly been frozen by flash cryocooling at ambient pressure, which often requires a time-consuming search for cryoprotection conditions. Recently, the CHTSB-affiliated team (led by Prof. Sol Gruner) at the Cornell High Energy Synchrotron Source (CHESS) has developed an alternative procedure, high-pressure cryocooling, which does not require the addition of chemical cryoprotectants. The result of high-pressure cryocooling is very often a dramatic improvement in the quality and resolution of the diffraction data as seen in Fig. 1.  The Cornell team is currently investigating the basic underlying principles and experimental parameters important for the optimization of the high pressure cooling method.

Since the high-pressure method involves the use of helium gas as a pressurizing medium, attention has been focused on its extension to diffraction phasing by incorporating heavy noble gases such as krypton or xenon. In the test case of Porcine Pancreatic Elastase (PPE, 240 residues, 26kDa), very high quality diffraction was obtained by the modified high-pressure cyrocooling method without the help of cryoprotectants. Furthermore, a single krypton site with an occupancy of 0.31 could be used successfully for SAD phasing at 1.3Å resolution (Fig. 2). The Cornell team is currently working on equipment modifications that will be compatible with a high throughput crystallography pipeline.

Fig. 2. A section of the electron density map for PPE contoured at 1s before density modification at 1.3Å resolution. The final refined model of PPE was superimposed for map evaluation. After density modification, a model-building program, ARP/wARP, could automatically trace 220 residues out of 240.