RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.

Mechanism of IFT-A polymerization into trains for ciliary transport.

Intraflagellar transport (IFT) is the highly conserved process by which proteins are transported along ciliary microtubules by a train-like polymeric assembly of IFT-A and IFT-B complexes. IFT-A is sandwiched between IFT-B and the ciliary membrane, consistent with its putative role in transporting transmembrane and membrane-associated cargoes. Here, we have used single-particle analysis electron cryomicroscopy (cryo-EM) to determine structures of native IFT-A complexes. We show that subcomplex rearrangements enable IFT-A to polymerize laterally on anterograde IFT trains, revealing a cooperative assembly mechanism. Surprisingly, we discover that binding of IFT-A to IFT-B shields the preferred lipid-binding interface from the ciliary membrane but orients an interconnected network of ß-propeller domains with the capacity to accommodate diverse cargoes toward the ciliary membrane. This work provides a mechanistic basis for understanding IFT-train assembly and cargo interactions.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology.

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes.

Structural Insights into Type III Secretion Systems of the Bacterial Flagellum and Injectisome.

Two of the most fascinating bacterial nanomachines-the broadly disseminated rotary flagellum at the heart of cellular motility and the eukaryotic cell-puncturing injectisome essential to specific pathogenic species-utilize at their core a conserved export machinery called the type III secretion system (T3SS). The T3SS not only secretes the components that self-assemble into their extracellular appendages but also, in the case of the injectisome, subsequently directly translocates modulating effector proteins from the bacterial cell into the infected host. The injectisome is thought to have evolved from the flagellum as a minimal secretory system lacking motility, with the subsequent acquisition of additional components tailored to its specialized role in manipulating eukaryotic hosts for pathogenic advantage. Both nanomachines have long been the focus of intense interest, but advances in structural and functional understanding have taken a significant step forward since 2015, facilitated by the revolutionary advances in cryo-electron microscopy technologies. With several seminal structures of each nanomachine now captured, we review here the molecular similarities and differences that underlie their diverse functions.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.

Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

DEMO-EM2: assembling protein complex structures from cryo-EM maps through intertwined chain and domain fitting.

The breakthrough in cryo-electron microscopy (cryo-EM) technology has led to an increasing number of density maps of biological macromolecules. However, constructing accurate protein complex atomic structures from cryo-EM maps remains a challenge. In this study, we extend our previously developed DEMO-EM to present DEMO-EM2, an automated method for constructing protein complex models from cryo-EM maps through an iterative assembly procedure intertwining chain- and domain-level matching and fitting for predicted chain models. The method was carefully evaluated on 27 cryo-electron tomography (cryo-ET) maps and 16 single-particle EM maps, where DEMO-EM2 models achieved an average TM-score of 0.92, outperforming those of state-of-the-art methods. The results demonstrate an efficient method that enables the rapid and reliable solution of challenging cryo-EM structure modeling problems.

The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

In situ architecture and membrane fusion of SARS-CoV-2 Delta variant.

Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the in situ structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant.

Knowledge Transfer from Macro-world to Micro-world: Enhancing 3D Cryo-ET Classification through Fine-Tuning Video-based Deep Models.

MOTIVATION: Deep learning models have achieved remarkable success in a wide range of natural-world tasks, such as vision, language, and speech recognition. These accomplishments are largely attributed to the availability of open-source large-scale datasets. More importantly, pre-trained foundational modellearnings exhibit a surprising degree of transferability to downstream tasks, enabling efficient learning even with limited training examples. However, the application of such natural-domain models to the domain of tiny Cryo-Electron Tomography (Cryo-ET) images has been a relatively unexplored frontier. This research is motivated by the intuition that 3D Cryo-ET voxel data can be conceptually viewed as a sequence of progressively evolving video frames. RESULTS: Leveraging the above insight, we propose a novel approach that involves the utilization of 3D models pre-trained on large-scale video datasets to enhance Cryo-ET subtomogram classification. Our experiments, conducted on both simulated and real Cryo-ET datasets, reveal compelling results. The use of video initialization not only demonstrates improvements in classification accuracy but also substantially reduces training costs. Further analyses provide additional evidence of the value of video initialization in enhancing subtomogram feature extraction. Additionally, we observe that video initialization yields similar positive effects when applied to medical 3D classification tasks, underscoring the potential of cross-domain knowledge transfer from video-based models to advance the state-of-the-art in a wide range of biological and medical data types. AVAILABILITY AND IMPLEMENTATION: https://github.com/xulabs/aitom.

Structures and Efflux Mechanisms of the AcrAB-TolC Pump.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.

In situ structure of intestinal apical surface reveals nanobristles on microvilli.

Microvilli are actin-bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause gastrointestinal disorders; however, mechanisms controlling microvilli formation and organization remain unresolved. Here, we study microvilli by vitrifying the Caenorhabditis elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them with cryo-focused ion-beam milling, followed by cryo-electron tomography and subtomogram averaging. We find that many radial nanometer bristles referred to as nanobristles project from the lateral surface of nematode and mouse microvilli. The C. elegans nanobristles are 37.5 nm long and 4.5 nm wide. Nanobristle formation requires a protocadherin family protein, CDH-8, in C. elegans. The loss of nanobristles in cdh-8 mutants slows down animal growth and ectopically increases the number of Y-shaped microvilli, the putative intermediate structures if microvilli split from tips. Our results reveal a potential role of nanobristles in separating microvilli and suggest that microvilli division may help generate nascent microvilli with uniformity.