Solution of protein crystallographic structures by high-pressure cryocooling and noble-gas phasing.
Room-pressure flash-cryocooling of protein crystals is the standard way to reduce radiation damage during data collection. Typically, it is necessary to find cryoprotection conditions by trial and error, a process that is not always successful. Recently, a new method, high-pressure cryocooling, was developed that does not require penetrative cryoprotectants and typically yields very high quality diffraction. Since this method involves helium gas as a pressurizing medium, it was of great interest to see whether the method could be extended to diffraction phasing by the incorporation of heavy noble gases such as krypton. A modified Kr-He high-pressure cyrocooling procedure is described wherein crystals are first pressurized with krypton gas to 10 MPa for 1 h. The krypton pressure is then released and the crystals are repressurized with helium over 150 MPa and cooled to liquid-nitrogen temperatures. Porcine pancreas elastase (PPE; 240 residues, 26 kDa) was selected as a test case for this study. Excellent diffraction was achieved by high-pressure cryocooling without penetrating cryoprotectants. A single 0.31 occupied krypton site in a PPE molecule [Bijvoet amplitude ratio (|DeltaF|/F) of 0.53%] was successfully used for SAD phasing at 1.3 A. This method has the potential to greatly simplify obtaining protein structures.
Kim
Chae Un
CU
Field of Biophysics, Cornell University, Ithaca, NY 14853, USA.
Hao
Quan
Q
Gruner
Sol M
SM
D000399:Air Pressure; D000818:Animals; D001665:Binding Sites; D003451:Cryoprotective Agents / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D005615:Freezing; D006371:Helium / Q000737:chemistry; D007726:Krypton / Q000737:chemistry; D008958:Models, Molecular; D005741:Noble Gases / Q000737:chemistry*; D010196:Pancreatic Elastase / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013552:Swine
D003451:Cryoprotective Agents; D005741:Noble Gases; D011506:Proteins; D006371:Helium; D007726:Krypton; D010196:Pancreatic Elastase