Summary The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) 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 Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.

Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) 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 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+.

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12 A by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.

An 11.7-Å-resolution cryo-EM map of the yeast 80S . eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcinricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S . eEF2 . sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.

Summary AMPA-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 signaling 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 elucidate the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.

Ca2+-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1–4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane, increasing significantly in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2)7,9. We previously solved crystal structures of detergent-solubilized rat TRPV6 in the closed state10,11. Corroborating previous electrophysiological studies3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that tightly binds Ca2+. However, it has remained unknown how TRPV6 channels open and close their pores for ion permeation. Here we present cryo-EM structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining S6 helices at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This novel gating mechanism, which defines the constitutive activity of TRPV6, is unique for tetrameric ion channels and provides new structural insights for understanding their diverse roles in physiology and disease.