Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca balance. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators.

Author Correction: Structure of human GABA receptor in an inactive state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of human GABA receptor in an inactive state.

The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA receptor has two subunits, GABA and GABA, that are structurally homologous but perform distinct and complementary functions. GABA recognizes orthosteric ligands, while GABA couples with G proteins.

Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP-activated channel TAX-4, reconstituted in lipid nanodiscs.

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species.

The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.

Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography.

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers.

Opening of the human epithelial calcium channel TRPV6.

Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues. Altered TRPV6 expression is associated with a variety of human diseases, including cancers. TRPV6 channels are constitutively active and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate.

Channel opening and gating mechanism in AMPA-subtype glutamate receptors.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength, and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist- and agonist-bound states and determine the iris-like mechanism of ion channel opening.

Structural Bases of Desensitization in AMPA Receptor-Auxiliary Subunit Complexes.

Fast excitatory neurotransmission is mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). AMPARs, localized at post-synaptic densities, are regulated by transmembrane auxiliary subunits that modulate AMPAR assembly, trafficking, gating, and pharmacology. Aberrancies in AMPAR-mediated signaling are associated with numerous neurological disorders.

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM.

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation.

Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.

Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process.

Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit.

Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM.

Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM.

With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit.

Structural Basis for Gating and Activation of RyR1.

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain.

Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy.

AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, gating, and pharmacology is tightly controlled by transmembrane AMPAR regulatory proteins (TARPs). Here, we used cryo-electron microscopy to elucidate the structural basis of AMPAR regulation by one of these auxiliary proteins, TARP γ2, or stargazin (STZ).

Structure of a mammalian ryanodine receptor.

Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.

Structure of the mammalian ribosomal pre-termination complex associated with eRF1.eRF3.GDPNP.

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition.

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit.

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown.

Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29.

Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments.

Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1.