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The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. A high-resolution electron cryo-microscopy structure of the zebrafish α1 glycine receptor bound to agonists or antagonists reveals the conformational changes that take place when the channel transitions from closed to open state. Glycine receptor mechanism Eric Gouaux and colleagues have determined the high-resolution electron cryo-microscopy structure of strychnine-sensitive glycine receptor (GlyR) from zebrafish, bound to agonists or antagonists to reveal the conformational changes that take place when the channel opens. GlyRs mediate neurotransmission throughout the spinal cord and brainstem and their dysfunction is linked to multiple neurological disorders, including autism and hyperekplexia. Also in this issue of Nature , Xin Huang et al . report the X-ray crystal structure of the human GlyR in the presence of the antagonist strychnine.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn’t show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain–heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein–protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis.

Comprehensive in situ structures of macromolecules can transform our understanding of biology and advance human health. Here, we map protein synthesis inside human cells in detail by combining automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo electron microscopy (cryo-EM). With this in situ cryo-EM approach, we resolved a 2.2 Å consensus structure of the human 80S ribosome and unveiled 23 functional states, nearly all better than 3 Å resolution. Compared to in vitro studies, we observed variations in ribosome structures, distinct environments of ion and polyamine binding, and associated proteins such as EDF1 and NAC β that are typically not enriched with purified ribosomes. We also detected additional peptide-related density features on the ribosome and visualized ribosome–ribosome interactions in helical polysomes. Finally, high-resolution structures from cells treated with homoharringtonine and cycloheximide revealed a distinct translational landscape and a spermidine that interacts with cycloheximide at the E site, one of the numerous polyamines that also bind native ribosomes. These results underscore the value of high-resolution in situ studies in the native environment. Understanding protein synthesis in its cellular context is essential. Here, authors apply in situ cryo-EM to reveal the human ribosome at 2.2 Å resolution, capture 23 states, and uncover drug-specific translation features in native cells.

The subnanometre-resolution electron cryomicroscopy structure of TmrAB, a heterodimeric ABC transport protein, in a nucleotide-free, inward-facing conformation, is determined. An ABC transporter at 8.2-Å resolution The ATP-binding cassette (ABC) transporter is implicated in a number of human diseases and is an important drug target. It is a small hetero-oligomeric protein presenting a challenge to structural biologists. Here Yifan Cheng and colleagues report the 8.2 Å resolution electron cryomicroscopy structure of TmrAB, a 135 kDa heterodimeric ABC transport protein, in a nucleotide-free, inward-facing conformation. The structure shows that the cytoplasmic nucleotide-binding domains of this ABC transporter are in contact with each other. Comparison with the structures of other ABC transporters in various states suggest that the cytoplasmic nucleotide-binding domains slide and rotate during the transition from the inward-facing to the outward-facing conformation. ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains 1 , 2 . ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases 3 , 4 . TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus ; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif 5 . Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.

Transition metal oxides (TMOs) have attracted extensive research attentions as promising electrocatalytic materials. Despite low cost and high stability, the electrocatalytic activity of TMOs still cannot satisfy the requirements of applications. Inspired by kinetics, the design of hollow porous structure is considered as a promising strategy to achieve superior electrocatalytic performance. In this work, cubic NiO hollow porous architecture (NiO HPA) was constructed through coordinating etching and precipitating (CEP) principle followed by post calcination. Being employed to detect glucose, NiO HPA electrode exhibits outstanding electrocatalytic activity in terms of high sensitivity (1323 μA mM −1  cm −2 ) and low detection limit (0.32 μM). The excellent electrocatalytic activity can be ascribed to large specific surface area (SSA), ordered diffusion channels, and accelerated electron transfer rate derived from the unique hollow porous features. The results demonstrate that the NiO HPA could have practical applications in the design of nonenzymatic glucose sensors. The construction of hollow porous architecture provides an effective nanoengineering strategy for high-performance electrocatalysts.

The Schizosaccharomyces pombe HP1 protein, Swi6, is shown to exist in an auto-inhibited state when unbound to chromatin, switching to a spreading-competent state upon binding to the HK9 methyl mark; disrupting this switch affects heterochromatin assembly and gene silencing. On–off switch for heterochromatin assembly Heterochromatin silences large regions of the genome and is crucial for many nuclear processes. Spreading of heterochromatin is mediated by HP1 proteins that assemble on histone H3K9-methylated chromatin. Here, Geeta Narlikar and colleagues use a variety of approaches to show that the fission yeast HP1 protein, Swi6, exists in an auto-inhibited state when not bound to chromatin. But upon binding to the H3K9 methyl mark and nucleosomal DNA, it switches to a state that is competent for spreading. Disruption of this switching disrupts heterochromatin assembly and gene silencing. A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions 1 , 2 , 3 , 4 , 5 , 6 . Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation 1 , 2 , 3 , 4 , 5 , 6 . How HP1 proteins assemble on methylated nucleosomal templates and how the HP1–nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6–nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo . These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo .

Fecal microbes play an important role in the survival and health of wild animals. Spotted hyena (Crocuta crocuta) is one of the representative carnivores in Africa. In this study, we examined the fecal microflora of spotted hyena by conducting high-throughput sequencing of the fecal microbial 16S rRNA gene V3–V4 high mutation region. The effects of age, sex, and feeding environment on the fecal microbiota of spotted hyenas were determined. The results showed that the core bacteria phyla of spotted hyenas fecal microbiota include Firmicutes (at an average relative abundance of 53.93%), Fusobacteria (19.56%), Bacteroidetes (11.40%), Actinobacteria (5.78%), and Proteobacteria (3.26%), etc. Age, gender, and feeding environment all had important effects on the fecal microbiota of spotted hyenas, among which feeding environment might be the most significant. The abundance of the Firmicutes in the adult group was significantly higher than that in the juvenile group, whereas the abundance of Fusobacteria, Bacteroidetes, and Proteobacteria were significantly lower than that in the juvenile group. The abundance of Lachnospiraceae and Ruminococcaceae in the female group was significantly higher than that in the male group. There were significant differences between the fecal microbial communities of Jinan group and Weihai group, and microbes from the phyla Firmicutes and Synergistetes were representative species associated with the difference.

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the \"target zone\", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Evolutionarily conserved SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF ( N -ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes. Using single-particle electron cryomicroscopy, several structures are reported which illuminate the mechanisms of action of the ATPase NSF that disassembles the SNARE complex into individual protein components. SNARE protein recycling mechanism In a variety of cellular processes — including neurotransmitter release, hormone release and vesicle trafficking — the evolutionarily conserved SNARE proteins form a complex that drives fusion between membranes of two cellular compartments. Once fusion occurs, these complexes are disassembled by the ATPase enzyme NSF and the SNAP adaptor proteins to recycle individual SNAREs for another round of membrane fusion. This study reports the use of single-particle electron cryomicroscopy to determine sub-nanometre to near-atomic resolution structures of NSF and the 20S complex. This paper reports the structures of full-length NSF in ATP- and ADP-bound states, and those of the roughly 660-kilodalton NSF/SNAP/SNARE (20S) super-complex involving two different SNARE complexes. The authors' data provide unprecedented details of the inner-workings of these essential molecular machines.