Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

We used cryo-electron tomography to visualize Rous Sarcoma Virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. CA reassembles into cores housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity. The virions in our data set range from ~ 105 to ~ 175 nm in diameter. Their cores are highly polymorphic. We observe angular cores, including some that are distinctively "coffin-shaped" for which we propose a novel fullerene geometry; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain less material, suggesting incomplete packaging. From the tomograms, we measured the surface areas of cores and hence their contents of CA subunits. From the virion diameters, we estimated their original complements of Gag. We find that RSV virions, like HIV, contain unassembled CA subunits and the fraction of CA that is assembled correlates with core type; angular cores incorporate ~ 80% of the available subunits and open-ended tubes, ~ 30%. The number of glycoprotein spikes is variable (~ 0 to 118) and also correlates with core type; virions with angular cores average 82 spikes, whereas those with tubular cores, 14 spikes. These observations imply that initiation of capsid assembly, in which interactions of spike endodomains with the Gag layer play a role, is a critical determinant of core morphology.

During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm. Poliovirus is the causative agent of poliomyelitis and the type member of the Enterovirus genus. As such, it shares structural and functional similarities with other members of the Picornaviridae family, including the coxsackievirus B viruses, which are associated with cardiomyopathies, central nervous system (CNS) infections, and diabetes; rhinoviruses, which are the most significant cause of the common cold; echoviruses, which can cause aseptic meningitis, gastroenteritis, and respiratory diseases; enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), which have recently caused epidemics in Asia of hand-foot-and-mouth disease, with a high frequency of central nervous system involvement and with high morbidity and mortality; and the foot-and-mouth disease virus, which causes devastating outbreaks of foot-and-mouth disease in livestock (1).Mature poliovirus (which sediments at 160S) is a spherical, nonenveloped virus, with a diameter of approximately 30 nm. Its capsid consists of 60 copies each of 4 proteins (VP1 to VP4) arranged on a Tϭ1 (pseudo-Tϭ3) icosahedral lattice and encloses a 7,500-base positive-sense single-stranded RNA (ssRNA) viral genome (2, 3). The major proteins (VP1, VP2, and VP3) share a topology, which is an eight-stranded beta barrel. Each protein has a different set of loops connecting the strands in the barrel and unique N-and C-terminal extensions. The C-terminal extensions of the VP1 to -3 subunits are located on the exterior surface of the virus, whereas VP4 and the N-terminal extensions are intertwined on the interior of the shell, making contact with the RNA genome. VP4, which is on the inside surface of the capsid, folds into an elongated structure, due to its contacts on the inner surfaces of the capsid protein that make up the shell. The outer surface of the virus is dominated by star-shaped mesas surrounding each 5...

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin’s structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin’s contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin’s chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.