Contraction of striated muscles is driven by cyclic interactions of myosin head projecting from the thick filament with actin filament and is regulated by Ca²⁺ released from sarcoplasmic reticulum. Muscle thin filament consists of actin, tropomyosin and troponin, and Ca²⁺ binding to troponin triggers conformational changes of troponin and tropomyosin to allow actin-myosin interactions. However, the structural changes involved in this regulatory mechanism remain unknown. Here we report the structures of human cardiac muscle thin filament in the absence and presence of Ca²⁺ by electron cryomicroscopy. Molecular models in the two states built based on available crystal structures reveal the structures of a C-terminal region of troponin I and an N-terminal region of troponin T in complex with the head-to-tail junction of tropomyosin together with the troponin core on actin filament. Structural changes of the thin filament upon Ca²⁺ binding now reveal the mechanism of Ca²⁺ regulation of muscle contraction.

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton-protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH-FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus.

Summary: Torque generation in the Salmonella flagellar motor is coupled to translocation of H⁺ ions through the proton‐conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan‐binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C‐terminal periplasmic fragment of MotB (MotB_C) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton‐conducting channel. Drastic conformational changes in the N‐terminal portion of MotB_C are required both for PG binding and the proton channel activation.

Construction of the bacterial flagellum in the cell exterior proceeds at its distal end by highly ordered self-assembly of many different component proteins, which are selectively exported through the central channel of the growing flagellum by the flagellar type III export apparatus. FliI is the ATPase of the export apparatus that drives the export process. Here we report the 2.4 Å resolution crystal structure of FliI in the ADP-bound form. FliI consists of three domains, and the whole structure shows extensive similarities to the α and β subunits of F ₀ F ₁ -ATPsynthase, a rotary motor that drives the chemical reaction of ATP synthesis. A hexamer model of FliI has been constructed based on the F ₁ -ATPase structure composed of the α ₃ β ₃ γ subunits. Although the regions that differ in conformation between FliI and the F ₁ -α/β subunits are all located on the outer surface of the hexamer ring, the main chain structures at the subunit interface and those surrounding the central channel of the ring are well conserved. These results imply an evolutionary relation between the flagellum and F ₀ F ₁ -ATPsynthase and a similarity in the mechanism between FliI and F ₁ -ATPase despite the apparently different functions of these proteins.

Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.