Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.DOI: http://dx.doi.org/10.7554/eLife.24905.001

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca2+ signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure–function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ~85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca2+-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

Tuberculosis continues to be a major disease threatening millions of lives worldwide. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. We have expressed and purified the 16-kDa antigen, an immunodominant antigen with serodiagnostic value, which has been previously cloned and shown to share low sequence homology with the ␣-crystallinrelated small heat shock protein family. Sedimentation equilibrium analytical ultracentrifugation and dynamic light scattering demonstrate the formation of a specific oligomer, 149 ؎ 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 ؎ 6 kDa (or trimer) is produced. Analysis by electron cryomicroscopy reveals a triangular shaped oligomeric structure arising from the presence of three subparticles or globules. Taken together, the data suggest an antigen complex structure of a trimer of trimers. This antigen, independent of ATP addition, effectively suppresses the thermal aggregation of citrate synthase at 40°C, indicating that it can function as a molecular chaperone in vitro. A complex between the antigen and heat-denatured citrate synthase can be detected and isolated using high performance liquid chromatography. We propose to rename the 16-kDa antigen Hsp16.3 to be consistent with other members of the small heat shock protein family.

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of fulllength rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-Å structure of TRPV1 determined by using singleparticle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N-and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.cryoelectron microscopy ͉ ion channels ͉ TRP channels

We exploit the random orientations of ice-embedded molecules imaged in an electron cryomicroscope to determine the three-dimensional structure of the Ca(2+)-release channel from the sarcoplasmic reticulum (SR) in its closed state, without tilting the specimen holder. Our new reconstruction approach includes an exhaustive search of all different characteristic projection images in the micrographs and the assignment of Euler angle orientations to these views. The 30 A map implied reveals a structure in which the transmembrane region exhibits no apparent opening on the SR lumen side. The extended cytoplasmic region has a hollow appearance and consists, in each monomer, of a clamp-shaped and a handle-shaped domain.

Here we present the determination of the three-dimensional structure of the skeletal muscle Ca2+-release channel in an open state using electron cryomicroscopy and angular reconstitution. In contrast to our reconstruction of the channel in its closed state, the density map of the channel driven towards its open state, by the presence of Ca2+ and ryanodine, features a central opening in the transmembrane region-the likely passageway for Ca2+ ions from the sarcoplasmic reticulum to the cytosol. The opening of the channel is associated with a 4 degree rotation of its transmembrane region with respect to its cytoplasmic region, and with significant mass translocations within the entire cytoplasmic region of the channel tetramer.

Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.

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