High-pressure cooling of protein crystals without cryoprotectants.
Flash-cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and in some cases unsuccessful. A method is described to cool protein crystals in high-pressure helium gas without the need for penetrative cryoprotectants. The method involves mounting protein crystals from the native mother liquor in a cryoloop with a droplet of oil, pressurizing the crystal to 200 MPa in He gas, cooling the crystal under pressure and then releasing the pressure. The crystal is then removed from the apparatus under liquid nitrogen and handled thereafter like a normal cryocooled crystal. Results are presented from three representative proteins. Dramatic improvement in diffraction quality in terms of resolution and mosaicity was observed in all cases. A mechanism for the pressure cooling is proposed involving high-density amorphous (HDA) ice which is produced at high pressure and is metastable at room pressure and 110 K.
Cornell High Energy Synchrotron Source (CHESS), USA.
Chae Un
CU
Kim
Raphael
R
Kapfer
Sol M
SM
Gruner
D017836:Agkistrodon / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000737:chemistry; D000594:Amino Acid Oxidoreductases / Q000737:chemistry; D000818:Animals; D003451:Cryoprotective Agents; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D005615:Freezing; D050830:L-Amino Acid Oxidase; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry; D011312:Pressure; D011506:Proteins / Q000737:chemistry*
D003451:Cryoprotective Agents; D010940:Plant Proteins; D011506:Proteins; C003427:thaumatin protein, plant; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase