Each cell of Salmonella typhimurium possesses a chemosensory apparatus, which detects gradients in its environment, and a flagellum, which propels the cell up or down the gradient. A set of five different proteins comprise a sensor. Three of these form a stable complex. The sensor regulates the phosporylation of the response regulator, which carries the signal from the sensor to the flagellum. The flagellum is the cell's propulsion system. It contains an 18,000 rpm, reversible rotary motor and an external propeller. The motor is powered directly by the proton motive gradient across the cell membrane. About 40 different proteins are required for the regulation, assembly and operation of the flagellum. Nineteen of the 40 proteins are known to be part of the assembled structure. There are likely to be a few more found in the final tally but these will be involved in the export/assembly pathway. Our goal is to determine the structures and mechanisms of these two remarkable machines. We will use electron cryomicroscopy to produce 3D molecular- resolution maps of the machines into which we can dock atomic models for the components. This approach is a frontier of structural biology. We propose to produce an atomic model for the cytoplasmic portion of a sensor, which contains the signaling domain of the transmembrane receptor, a kinase, and an adapter molecule. The entire complex has a mass of 1.4 106 daltons, consisting of 28 copies of TarC (the cytoplasmic domain of the receptor), 6 copies of CheW (adapter protein), and 4 copies of CheA (histidine kinase). The atomic structures for TarC and most of CheA are known. The structure of CheW can be modeled because of its homology to CheA. We have good preliminary data on the whole complex, which augers well for completion of a 20Angstrom units map of the complex. We propose to obtain 3D maps at molecular resolution of the rotor of the flagellum. Our preparations retain three of the key torque-generating, direction-switching elements. They lack the transmembrane proton channel proteins. We plan to compare maps from clockwise-turning motors and from counterclockwise-turning motors. We plan to obtain maps with the response regulator bound. These maps will give insights into the motor's mechanism of torque generation and switching. Only one domain of one of the key proteins is available at atomic resolution, so an atomic model for the motor remains in the future. We propose to obtain 3D maps of the propeller apparatus (hook and filament) at about 4 Angstrom units resolution by electron cryomicroscopy. These maps should permit us to produce a chain tracing of the peptides.
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| Dailey, Frank E; Macnab, Robert M (2002) Effects of lipoprotein biogenesis mutations on flagellar assembly in Salmonella. J Bacteriol 184:771-6 |
| Li, Huilin; DeRosier, David J; Nicholson, William V et al. (2002) Microtubule structure at 8 A resolution. Structure 10:1317-28 |
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