High pressure cryocooling (Hi-P cryo) is a crystal handling technique that has been developed at Cornell University that can often yield high quality diffraction from cryocooled crystals (1). However, it is NOT a magic bullet. To see whether Hi-P cryo will help you, please read the information below and complete the checklist that follows.
Hi-P cryo is worth trying if you have crystals that diffract well at room temperature, but have problems when cryocooled by normal means. These problems include degraded diffraction quality of crystals cryocooled in the usual way (i. e. with added cryprotectant), as well as inability to find any suitable cryoprotectant after exhaustive search. Hi-P cryo has been is gaining attention and has been successfully implemented at several facilities (2, 3), aside from Cornell and CHESS.
Hi-P cryo will not generally make crystals that diffract poorly at room temperature diffract better. If your crystals do not diffract well to begin with, we do not advise trying to Hi-P cool them.
In a typical Hi-P cryo experiment, the crystals are coated with an oil, such as Hampton Paratone oil. The crystal is then picked up in a cryoloop with a minimal oil droplet, pressurized to 2000 atmospheres in helium gas and cryocooled while at high pressure. The pressure is then released; as long as the crystals are kept cold they can be handled, and diffraction data collected from them, at room pressure, just as a normal cryocooled crystal. The crystals will irreversibly degrade if warmed above about 130k.
Routine Hi-P cryo has limitations: The crystals must be capable of being transferred into oil. Problematic situations that argue against Hi-P cryo include crystals that are degraded by oil, or are unusually delicate, or simply too small (less than about 100 microns across) to be easily handled.
Although Hi-P cryo is most often used for crystals that have problems with routine cryocooling, there are other specialized applications. These include use of Hi-P cryo to produce Xe or Kr derivatized crystals and to induce repacking of virus crystals. Methods that do not use oil coating have been developed, though these are not routine. Users interested in these more specialized applications are advised to consult the literature (1-14) and then to contact us for further discussions.
- Kim CU, Kapfer R, Gruner SM. 2005. High-pressure cooling of protein crystals without cryoprotectants. Acta Crystallographica Section D-Biological Crystallography 61: 881-90
- Higashiura A, Ohta K, Masaki M, Sato M, Inaka K, et al. 2013. High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method. Journal of Synchrotron Radiation 20: 989-93
- van der Linden P, Dobias F, Vitoux H, Kapp U, Jacobs J, et al. 2014. Towards a high-throughput system for high-pressure cooling of cryoprotectant-free biological crystals. Journal of Applied Crystallography 47: 584-92
- Albright RA, Ibar J-LV, Kim CU, Gruner SM, Morais-Cabral JH. 2006. The RCK domain of the KtrAB K+ transporter: Multiple conformations of an octameric ring. Cell 126: 1147-59
- Avvaru BS, Kim CU, Sippel KH, Gruner SM, Agbandje-McKenna M, et al. 2010. A Short, Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II. Biochemistry 49: 249-51
- Domsic JF, Avvaru BS, Kim CU, Gruner SM, Agbandje-McKenna M, et al. 2008. Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II. Journal of Biological Chemistry 283: 30766-71
- Fourme R, Ascone I, Kahn R, Mezouar M, Bouvier P, et al. 2002. Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography - Technical advance. Structure 10: 1409-14
- Kim CU, Barstow B, Tate MW, Gruner SM. 2009. Evidence for liquid water during the high- density to low-density amorphous ice transition. Proceedings of the National Academy of Sciences of the United States of America 106: 4596-600
- Kim CU, Chen Y-F, Tate MW, Gruner SM. 2008. Pressure-induced protein crystals. Journal of Applied Crystallography 41: 1-7
- Kim CU, Hao Q, Gruner SM. 2006. Solution of protein crystallographic structures by high- pressure cryocooling and noble-gas phasing. Acta Crystallographica Section D-Biological Crystallography 62: 687-94
- Kim CU, Hao Q, Gruner SM. 2007. High-pressure cryocooling for capillary sample cryoprotection and diffraction phasing at long wavelengths. Crystallography 63: 653-59
- Kim CU, Tate MW, Gruner SM. 2011. Protein dynamical transition at 110 K. Proceedings of the National Academy of Sciences of the United States of America 108: 20897-901
- Kim CU, Wierman JL, Gillilan R, Lima E, Gruner SM. 2013. A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter. Journal of Applied Crystallography 46: 234-41
- Toms AV, Deshpande A, McNally R, Jeong Y, Rogers JM, et al. 2013. Structure of a pseudokinase- domain switch that controls oncogenic activation of Jak kinases. Nature Structural & Molecular Biology 20: 1221-23