X-ray diffraction data is the main source of 3-dimensional structure information at atomic resolution for proteins. While a great deal of information is potentially available from this technique, its application requires the ability to prepare crystals of size and order suitable for X-ray analysis. This has proved especially difficult for membrane proteins. Cytochrome reductase (the cytochrome bc1 complex) is a membrane protein complex which makes up the middle segment of the mitochondrial respiratory chain. The respiratory chain is responsible for biological oxidation and for conservation of the energy released in the form of a proton electrochemical potential gradient across the mitochondrial inner membrane. Energy from this gradient is then used to synthesize ATP or to do work by transporting substances across the membrane. A number of mitochondrial myopathies have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in cytochrome reductase. The drug atovaquinone used to control secondary infections in aids patients, is probably an inhibitor of the cytochrome reductase. Several important crop protection fungicides are reductase inhibitors. We have solved the structure of the bc1 complex by isomorphous replacement in the chicken (P212121) crystals and molecular replacement in the hexagonal (P6522) crystals from rabbit and beef heart and the monoclinic (P21) crystals from beef heart. The Rieske protein occupies different positions in different crystals, contacting cytochrome c1 in the native beef crystals and the presumed Qo site under the PEWY loop of cytochrome b in the presence of the quinoid inhibitor stigmatellin. The model is being refined against datasets from native and inhibitor- complexed crystals. It is proposed to (a) Complete phase improvement, model building and refinement to determine atomic coordinates at intermediate resolution using the present data, and (b) further improve the crystals to diffract at 2.5 Angstrom units or better with good completeness, so we can investigate subtle features (such as substrate/inhibitor binding, hydrogen bonding interactions, salt bridges, and bound solvent).
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