Crystallization and preliminary crystallographic analysis of the Rhodobacter capsulatus bc1 complex.

L.-S. Huang, D. Cobessi,M. Ban, N.G. Pon, F. Daldal, E.A. Berry


The cytochrome bc1 complex was purified by ion exchange and gel-filtration chromatography from the over-producing strain of Rhodobacter capsulatus  (pMTS1/MT-RBC1). Crystallization attempts yielded three different crystal forms, all small and relatively disordered. Slightly different variants of the P21 form were obtained in the presence and absence of stigmatellin. Four data sets complete to 6 or 7 Å were collected and phased by MR using a chicken-based homology model. Multi-crystal and NCS averaging were used to improve the MR phases and eliminate model bias. Although little detail can be seen at this resolution, the solutions indicate that the crystals contain all three subunits and hold out the promise for more structural information as soon as the crystalline order can be improved.

1. Diffraction pattern of P21 crystal of Rhodobacter capsulatus bc1 complex. The image was collected during a 1-degree oscillation at cryogenic temperature. While useful data extends to only about 6.5 Å,

dark rows of scattering indicate some order as high as 4 Å.

2. Packing arrangement in the P21 crystals. The assymetric unit contains a dimer with the 2-fold axis oriented in the ab plane nearly perpendicular to a. This crystal form can be made in the presence or absence of stigmatellin, with some variation in cell parameters which may or may not be correlated.

3. Packing arrangement in the P3(1)12 crystals. The dimer 2-fold axis aligns with a crystallographic 2-fold, giving a monomer in the asymmetric unit.

4. Packing arrangement in the P4(2)2(1)2 crystals. Again the dimer 2-fold axis aligns with a crystallographic 2-fold, giving a monomer in the asymmetric unit.

5,6. Electron Density obtained by phasing four different crystal forms (two were non-isomorphous P21 crystals) with the molecular replacement models, then performing 16 cycles of NCS/multicrystal averaging, then taking the averaged density from the last cycle. In each case the backbone diagrams are the molecular replacement model (based on the mitochondrial bc1 structure) and the electron density maps (blue webs) are the density from the Rhodobacter crystals. The maps are contoured at 3 sigma to show the main features, more detail is avalable by contouring at a lower level. (5) is the view looking down on the P-side surface, i.e. the point of view of cytochrome c2. (6) is a section of the transmembrane helices containing most of cytochrome b.