title: Preliminary cryocrystallographic study of the mitochondrial cytochrome bc1 complex: improved crystallization and flash-cooling of a large membrane protein.
abstract: Ubiquinol-cytochrome c reductase is a crucial integral membrane protein in the mitochondrial respiratory cycle. Eleven subunits containing three cytochrome heme groups and a 2Fe-2S Rieske center make up this 240 kDa enzyme complex. Previously, many different crystal forms of the bc1 complex have displayed diffraction to as far as 4.5 A. However, rapid degradation of the protein in the X-ray beam at room temperature has obstructed the collection of a full data set from a single crystal. As slight heterogeneities between crystals severely hampered merging of data from different crystals, we sought a method to stabilize the protein crystal in the X-ray beam in order to collect a full data set from one crystal sample. To this end, water soluble protein crystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In this communication, we report on a successful experiment in which flash-cooled bc1 membrane protein crystals have given rise to sustained diffraction over a 60 hour data collection period at a synchrotron source. Furthermore, we present an improved purification and crystallization protocol yielding crystals readily diffracting out to 3.3 A. These results should greatly aid in the future realization of the molecular structure of the bc1 complex as well as other membrane proteins.
authors.affiliation: Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Chan
authors.forename: T V
authors.initials: TV
authors.lastname: Law
authors.forename: H J
authors.initials: HJ
authors.lastname: Kwon
authors.forename: B K
authors.initials: BK
authors.lastname: Jap
mesh_terms: D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D014450:Electron Transport Complex III / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000648:ultrastructure*; D008929:Mitochondria, Heart
chemical_list: D008565:Membrane Proteins; D014450:Electron Transport Complex III