Structure and inhibition of SARS-CoV-2 spike refolding in membranes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds the receptor angiotensin converting enzyme 2 (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas the S1 domain contains high sequence variability, the S2 domain is conserved and is a promising pan-betacoronavirus vaccine target. We applied cryo-electron tomography to capture intermediates of S2 refolding and understand inhibition by antibodies to the S2 stem-helix.

Visualizing the translation landscape in human cells at high resolution.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and single-particle cryo-electron microscopy (cryo-EM). With this cryo-EM approach we resolved a 2.

Therapeutic effect of Wendan Decoction combined with mosapride on gastroesophageal reflux disease after esophageal cancer surgery.

Background: Gastroesophageal reflux disease (GERD) is a common complication of esophageal cancer surgery that can affect quality of life and increase the risk of esophageal stricture and anastomotic leakage. Wendan Decoction (WDD) is a traditional Chinese herbal formula used to treat various gastrointestinal disorders, such as gastritis, functional dyspepsia, and irritable bowel syndrome. Mosapride, a prokinetic agent, functions as a selective 5-hydroxytryptamine 4 agonist, enhancing gastrointestinal motility.

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM.

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20-30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.

Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA.

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome.

Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.

Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity.

Age, Gender, and Feeding Environment Influence Fecal Microbial Diversity in Spotted Hyenas (Crocuta crocuta).

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.

Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly.

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.

Cryo-EM structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome.

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-BeF that capture two potential reaction intermediates.

Cryo-EM structure of the αvβ8 integrin reveals a mechanism for stabilizing integrin extension.

Integrins are conformationally flexible cell surface receptors that survey the extracellular environment for their cognate ligands. Interactions with ligands are thought to be linked to global structural rearrangements involving transitions between bent, extended-closed and extended-open forms. Thus far, structural details are lacking for integrins in the extended conformations due to extensive flexibility between the headpiece and legs in this conformation.

Sensitive Nonenzymatic Electrochemical Glucose Detection Based on Hollow Porous NiO.

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.

CoO based non-enzymatic glucose sensor with high sensitivity and reliable stability derived from hollow hierarchical architecture.

Inspired by kinetics, the design of hollow hierarchical electrocatalysts through large-scale integration of building blocks is recognized as an effective approach to the achievement of superior electrocatalytic performance. In this work, a hollow, hierarchical CoO architecture (CoO HHA) was constructed using a coordinated etching and precipitation (CEP) method followed by calcination. The resulting CoO HHA electrode exhibited excellent electrocatalytic activity in terms of high sensitivity (839.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Electron cryo-microscopy structure of the mechanotransduction channel NOMPC.

Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels.

Single-particle cryo-EM data acquisition by using direct electron detection camera.

Recent advances in single-particle electron cryo-microscopy (cryo-EM) were largely facilitated by the application of direct electron detection cameras. These cameras feature not only a significant improvement in detective quantum efficiency but also a high frame rate that enables images to be acquired as 'movies' made of stacks of many frames. In this review, we discuss how the applications of direct electron detection cameras in cryo-EM have changed the way the data are acquired.

Glycine receptor mechanism elucidated by electron cryo-microscopy.

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).

Mechanistic insights into the recycling machine of the SNARE complex.

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.

Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter.

ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. 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. 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.

Selective targeting of TGF-β activation to treat fibroinflammatory airway disease.

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation.

A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly.

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. 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. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood.

Electron tomography of paracrystalline 2D arrays.

Paracrystalline arrays possess specific types of disorder that reduce the structural information as well as resolution when spatially averaged over repeating motifs. Electron tomography combined with motif classification and averaging can solve the heterogeneity problem and provide information on the structural elements that give rise to the disorder. This chapter describes procedures that would be used in a typical tomography application to identify and characterize a paracrystalline specimen.

Self-assembly of filamentous amelogenin requires calcium and phosphate: from dimers via nanoribbons to fibrils.

Enamel matrix self-assembly has long been suggested as the driving force behind aligned nanofibrous hydroxyapatite formation. We tested if amelogenin, the main enamel matrix protein, can self-assemble into ribbon-like structures in physiologic solutions. Ribbons 17 nm wide were observed to grow several micrometers in length, requiring calcium, phosphate, and pH 4.

Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation.

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.

Fabs enable single particle cryoEM studies of small proteins.

In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment.

Self-assembly of amelogenin proteins at the water-oil interface.

Self-assembly of amelogenin plays a key role in controlling enamel biomineralization. Recently, we generated self-aligning nanoribbons of amelogenin in water-in-oil emulsions stabilized by the full-length protein (rH174). Here, we tested the hypothesis that the hydrophilic C-terminus is critical for self-assembly of amelogenin into nanoribbons.