title: Solution of protein crystallographic structures by high-pressure cryocooling and noble-gas phasing.
abstract: 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.
authors.affiliation: Field of Biophysics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Chae Un
authors.initials: CU
authors.lastname: Kim
authors.forename: Quan
authors.initials: Q
authors.lastname: Hao
authors.forename: Sol M
authors.initials: SM
authors.lastname: Gruner
mesh_terms: 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
chemical_list: D003451:Cryoprotective Agents; D005741:Noble Gases; D011506:Proteins; D006371:Helium; D007726:Krypton; D010196:Pancreatic Elastase