PRELIMINARY CRYOCRYSTALLOGRAPHIC STUDY OF THE MITOCHONDRIAL CYTOCHROME BC(1) COMPLEX - IMPROVED CRYSTALLIZATION AND FLASH-COOLING OF A LARGE MEMBRANE-PROTEIN

Ubiquinol-cytochrome c reductase is a crucial integral membrane protei n in the mitochondrial respiratory cycle. Eleven subunits containing t hree cytochrome heme groups and a 2Fe-2S Rieske center make up this 24 0 kDa enzyme complex. Previously, many different crystal forms of the bc(1) complex have displayed diffraction to as far as 4.5 Angstrom. Ho wever, rapid degradation of the protein in the X-ray beam at room temp erature 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 stabil ize the protein crystal in the X-ray beam in order to collect a full d ata set from one crystal sample. To this end, water soluble protein cr ystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In t his communication, we report on a successful experiment in which flash -cooled bc(1) membrane protein crystals have given rise to sustained d iffraction over a 60 hour data collection period at a synchrotron sour ce. Furthermore, we present an improved purification and crystallizati on protocol yielding crystals readily diffracting out to 3.3 Angstrom. These results should greatly aid in the future realization of the mol ecular structure of the bc(1) complex as well as other membrane protei ns. (C) 1995 Academic Press Limited