Contractile System Research Articles

Archaeal type six secretion system mediates contact-dependent antagonism.

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

Abstract 4136116: Metformin Therapy for Reperfusion Injury Consolidates Cardioprotection in the High-Risk Elderly Heart

Introduction: Metformin (MET), a first-line diabetes therapy, exhibits cardioprotective effects during acute ischemia and reperfusion (I/R). Cardiac injury is enhanced in the aging heart in murine and humans. Acute, high dose MET treatment at the onset of reperfusion (R) markedly reduced infarct size (MI) in the high risk aged heart via modulation of mitochondrial complex I. However, the effect of subsequent chronic, standard dose MET to consolidate and possibly enhance cardioprotection and survival following I/R in the aged heart remains unknown. Hypothesis: Administration of chronic standard dose of MET will ameliorate mortality and morbidity following MI through rescuing an impaired adaptive metabolic remodeling. Methods: Aged (22-24 month) C57BL6/J mice were subjected to sham operations or 45 min of ischemia by LAD ligation followed by R. All mice received acute, high dose MET (2 mM, i.v.) at the onset of R followed by randomization to chronic MET (300 mg/kg/day) in drinking water versus vehicle (VEH) administered for 14 days. Cardiac function was determined by echocardiography weekly. IonOptix Myocyte Calcium and Contractility System measured the contractile functions and transient calcium signaling of isolated cardiomyocytes. Results: The Kaplan-Meier survival analysis showed that chronic MET treatment following acute treatment at the onset of R significantly reduced mortality compared to VEH group. Although chronic MET treatment did not further reduce MI size, echo and histology showed that chronic MET preserved diastolic function, prevented left ventricular hypertrophy and reduced collagen deposition following I/R in aging. Moreover, isolated cardiomyocytes from MET treated hearts showed improvements in contractile function, transit calcium signaling, and the coupling between contractility and calcium levels as compared to the cardiomyocytes from VEH group hearts. Immunoblotting results showed that Sens2/AMPK metabolic signaling was significantly upregulated by chronic MET post-MI, indicating the metabolic homeostasis modulated by MET is a critical factor involved in the beneficial effects of chronic MET on limiting I/R-induced myocardial damage that leads to deleterious remodeling in aging. Conclusions: The mortality and morbidity of MI in the high risk aged heart is attenuated with acute, high dose of MET treatment followed by chronic standard dose MET treatment to consolidate protection via rescuing the impaired adaptive metabolic response in aging.

Exploring heterogeneous cell population dynamics in different microenvironments by novel analytical strategy based on images.

Understanding the dynamic states and transitions of heterogeneous cell populations is crucial for addressing fundamental biological questions. High-content imaging provides rich datasets, but it remains increasingly difficult to integrate and annotate high-dimensional and time-resolved datasets to profile heterogeneous cell population dynamics in different microenvironments. Using hepatic stellate cells (HSCs) LX-2 as model, we proposed a novel analytical strategy for image-based integration and annotation to profile dynamics of heterogeneous cell populations in 2D/3D microenvironments. High-dimensional features were extracted from extensive image datasets, and cellular states were identified based on feature profiles. Time-series clustering revealed distinct temporal patterns of cell shape and actin cytoskeleton reorganization. We found LX-2 showed more complex membrane dynamics and contractile systems with an M-shaped actin compactness trend in 3D culture, while they displayed rapid spreading in early 2D culture. This image-based integration and annotation strategy enhances our understanding of HSCs heterogeneity and dynamics in complex extracellular microenvironments.

Unbiased high-throughput screening of drug-repurposing libraries identifies small-molecule inhibitors of clot retraction

AbstractPlatelet clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional αIIbβ3 receptors, fibrin, and the integrated actions of the actin-myosin contractile and cytoskeletal systems. Disturbances in clot retraction have been associated with both bleeding and thrombosis. We recently demonstrated that platelets treated with the αIIbβ3 antagonist peptide RGDW, which eliminates fibrinogen-mediated platelet aggregation, are still able to retract clots. We have exploited this observation to develop an unbiased, functional high-throughput assay to identify small-molecule inhibitors of fibrin-mediated clot retraction adapted for a 384-well plate format. We tested 9710 compounds from drug-repurposing libraries (DRLs). These libraries contain compounds that are either US Food and Drug Administration approved or have undergone preclinical/clinical development. We identified 27 compounds from the Library of Pharmacologically Active Compounds library as inhibitors of clot retraction, of which 14 are known inhibitors of platelet function. From the DRLs, we identified 135 compounds (1.6% hit rate). After extensive curation, these compounds were categorized based on the activity of their reported target. Multiple kinase and phosphodiesterase inhibitors with known antiplatelet effects were identified, along with multiple deubiquitination and receptor inhibitors, as well as compounds that have not previously been reported to have antiplatelet activity. Studies of 1 of the deubiquitination inhibitors (degrasyn) suggest that its effects are downstream of thrombin-induced platelet-fibrinogen interactions and thus may permit the separation of platelet thrombin-induced aggregation-mediated events from clot retraction. Additional studies of the identified compounds may lead to novel mechanisms of inhibiting thrombosis.

The Pvc15 ATPase selectively associates effector proteins with the Photorhabdus virulence cassette.

The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.

A study on the motion characteristics of particle in shear flow of active nematic fluid between two plates

Abstract The motion characteristics of particle in shear flow of active nematic fluid between two plates are investigated numerically. The influence of activity intensity of active nematic fluids on the motion characteristics of particle is focused. The results showed that, when the activity intensity is high, the particle exhibits initially smooth trajectories and ultimately approximate Brownian motion. As the activity intensity increases, both the translational mean square displacement (MSDT) and rotational mean square displacement (MSDR) of the particle increase. The particle exhibits initially super-diffusive behavior and then normal diffusion. The unique vortex lattice mode in the flow plays a role in hindering particle motion, leading to a decrease in MSDT and MSDR. An increase in particle size will lead to a weakening of particle diffusion. In addition, there are topological defects in the active nematic fluid, and the number of topological defects N def in the contractile system is greater than that in the extensible system, while the presence of particle has a smaller impact on N def in the flow.

Identification of novel toxins associated with the extracellular contractile injection system using machine learning

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

Contractile injection systems facilitate sporogenic differentiation of Streptomyces davawensis through the action of a phage tapemeasure protein-related effector

Contractile injection systems (CISs) are prokaryotic phage tail-like nanostructures loading effector proteins that mediate various biological processes. Although CIS functions have been diversified through evolution and hold the great potential as protein delivery systems, the functional characterisation of CISs and their effectors is currently limited to a few CIS lineages. Here, we show that the CISs of Streptomyces davawensis belong to a unique group of bacterial CISs distributed across distant phyla and facilitate sporogenic differentiation of this bacterium. CIS loss results in decreases in extracellular DNA release, biomass accumulation, and spore formation in S. davawensis. CISs load an effector, which is a remote homolog of phage tapemeasure proteins, and its C-terminal domain has endonuclease activity responsible for the CIS-associated phenotypes. Our findings illustrate that CISs can contribute to the reproduction of bacteria through the action of the effector and suggest an evolutionary link between CIS effectors and viral cargos.

Sarcomere. The structural unit of the myofibrillar complex of typical cardiomyocytes. Isometric aspects of sarcomere organization

Background. The complexity of understanding the structure and function of cardiomyocytes in the myocardium is a fundamental problem in the field of heart morphology. Sarcomeres are the main structural and functional units of myofibrils. Understanding the organization and dynamics of sarcomeres in cardiomyocytes is crucial for establishing the mechanisms responsible for the process of forming the contractile system of cardiomyocytes. Objective. The main purpose of this study is a review of literary sources that complement and expand the essential findings about the construction of myofibrils and their components when studied by effective methods of microscopic techniques. Materials. Literature review databases, systematic search of primary sources related to the research topic. Methods of visual examination of the components of sarcomeres and elements of their organization. Results. Transmission electron microscopy provides high-resolution imaging of sarcomeres, which is necessary for detailed analysis of their ultrastructure, including visualization of sarcomere components such as Z-discs, M-lines, thick and thin filaments. Morphometric methods are crucial for studying the compaction and orientation of myofibrils in cardiomyocytes. Conclusion. Morphometric methods provide valuable information about the structural remodeling of myofibrils in response to physiological stimuli or pathological conditions. These techniques are needed to improve our understanding of the mechanisms underlying muscle contraction and to develop methods to study cardiac remodeling processes in response to the adaptive response.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins.

Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.

Rhizoctonia solani disease suppression: addition of keratin-rich soil amendment leads to functional shifts in soil microbial communities.

Promoting soil suppressiveness against soil borne pathogens could be a promising strategy to manage crop diseases. One way to increase the suppression potential in agricultural soils is via the addition of organic amendments. This microbe-mediated phenomenon, although not fully understood, prompted our study to explore the microbial taxa and functional properties associated with Rhizoctonia solani disease suppression in sugar beet seedlings after amending soil with a keratin-rich waste stream. Soil samples were analyzed using shotgun metagenomics sequencing. Results showed that both amended soils were enriched in bacterial families found in disease suppressive soils before, indicating that the amendment of keratin-rich material can support the transformation into a suppressive soil. On a functional level, genes encoding keratinolytic enzymes were found to be abundant in the keratin-amended samples. Proteins enriched in amended soils were those potentially involved in the production of secondary metabolites/antibiotics, motility, keratin-degradation, and contractile secretion system proteins. We hypothesize these taxa contribute to the amendment-induced suppression effect due to their genomic potential to produce antibiotics, secrete effectors via the contractile secretion system, and degrade oxalate-a potential virulence factor of R. solani-while simultaneously possessing the ability to metabolize keratin.

Effect of aging on the human myometrium at single-cell resolution

Age-associated myometrial dysfunction can prompt complications during pregnancy and labor, which is one of the factors contributing to the 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we have constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty perimenopausal and postmenopausal women. We identify 23 myometrial cell subpopulations, including contractile and venous capillary cells as well as immune-modulated fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observe altered myometrial cell-to-cell communication as an aging hallmark, which associated with the loss of 25 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older individuals during pregnancy and labor.

Optimizing Cardioplegic Solutions: A Focus on Pharmaco-Cold Strategies

This study investigates the impact of crystalloid cardioplegic solutions (CCS) on electrophysiological mechanisms and the contractile system during the diastolic phase. Extracellular solutions, categorized by ion concentration, demonstrate efficacy in inducing cardiac arrest through moderately elevated potassium or potassium-magnesium combinations, aligning with normal or slightly reduced sodium and calcium levels. Notably, the ease of equilibration with myocardial tissue enhances the protective effect, correlating with infusion volume and duration of action. However, drawbacks such as low buffering capacity and the need for reinfusion after 15-20 minutes are identified. This research sheds light on the dynamic relationship between solution composition, duration of action, and protective efficacy, providing valuable insights for refining cardioplegic protocols in cardiac surgeries. Highlights: Intracellular vs. Extracellular Impact: Explore ion concentration's influence on crystalloid cardioplegic solutions, emphasizing the choice between intracellular and extracellular formulations. Equilibration Dynamics: Investigate the direct correlation between infusion volume, duration of action, and the protective effect, emphasizing equilibration with myocardial tissue. Refining Cardioplegic Protocols: Identify limitations like low buffering capacity, necessitating reinfusion, and offer insights into strategies for enhancing cardioplegic protocols in cardiac surgeries. Keywords: Cardioplegia, Intracellular Solutions, Calcium paradox, Protective Efficacy, Cellular Metabolism

An extensive disulfide bond network prevents tail contraction in Agrobacteriumtumefaciens phage Milano

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Controlling wall-particle interactions with activity.

We theoretically determine the effective forces on hard disks near walls embedded inside active nematic liquid crystals. When the disks are sufficiently close to the wall and the flows are sufficiently slow, we can obtain exact expressions for the effective forces. We find these forces and the dynamics of disks near the wall depend both on the properties of the active nematic and on the anchoring conditions on the disks and the wall. Our results show that the presence of active stresses attract planar anchored disks to walls if the activity is extensile, and repel them if contractile. For normal anchored disks the reverse is true; they are attracted in contractile systems, and repelled in extensile ones. By choosing the activity and anchoring, these effects may be helpful in controlling the self assembly of active nematic colloids.

Evolutionary and ecological role of extracellular contractile injection systems: from threat to weapon.

Contractile injection systems (CISs) are phage tail-related structures that are encoded in many bacterial genomes. These devices encompass the cell-based type VI secretion systems (T6SSs) as well as extracellular CISs (eCISs). The eCISs comprise the R-tailocins produced by various bacterial species as well as related phage tail-like structures such as the antifeeding prophages (Afps) of Serratia entomophila, the Photorhabdus virulence cassettes (PVCs), and the metamorphosis-associated contractile structures (MACs) of Pseudoalteromonas luteoviolacea. These contractile structures are released into the extracellular environment upon suicidal lysis of the producer cell and play important roles in bacterial ecology and evolution. In this review, we specifically portray the eCISs with a focus on the R-tailocins, sketch the history of their discovery and provide insights into their evolution within the bacterial host, their structures and how they are assembled and released. We then highlight ecological and evolutionary roles of eCISs and conceptualize how they can influence and shape bacterial communities. Finally, we point to their potential for biotechnological applications in medicine and agriculture.

The tip protein PAAR is required for the function of the type VI secretion system.

Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. However, the importance and the function of the tip protein PAAR in the T6SS remain unclear. Here, we characterized the PAAR protein of EAEC using biochemical, fluorescence microscopy, and antibacterial competition approaches. Using pull-down assays and cryo-electron microscopy analysis of the (VgrG)3-(Tle1)3-PAAR complex, we show that PAAR tops and closes the β-prism structure of the VgrG spike. The PAAR protein structure is further tightened by the zinc atom coordinated via conserved residues essential for its function. We provide evidence that PAAR is necessary for T6SS-1-mediated killing due to its requirement for proper T6SS baseplate assembly and further sheath polymerization. Our results suggest that the PAAR protein is an essential component of T6SS. IMPORTANCE The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

Bulk and single-cell alternative splicing analyses reveal roles of TRA2B in myogenic differentiation.

Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that occur during myogenesis are not well understood. Here, we employed isoform long-reads RNA-seq (Iso-seq) and single-cell RNA-seq (scRNA-seq) to investigate the AS landscape during myogenesis. Our Iso-seq data identified 61,146 full-length isoforms representing 11,682 expressed genes, of which over 52% were novel. We identified 38,022 AS events, with most of these events altering coding sequences and exhibiting stage-specific splicing patterns. We identified AS dynamics in different types of muscle cells through scRNA-seq analysis, revealing genes essential for the contractile muscle system and cytoskeleton that undergo differential splicing across cell types. Gene-splicing analysis demonstrated that AS acts as a regulator, independent of changes in overall gene expression. Two isoforms of splicing factor TRA2B play distinct roles in myogenic differentiation by triggering AS of TGFBR2 to regulate canonical TGF-β signalling cascades differently. Our study provides a valuable transcriptome resource for myogenesis and reveals the complexity of AS and its regulation during myogenesis.

Structure of Vibrio Phage XM1, a Simple Contractile DNA Injection Machine.

Antibiotic resistance poses a growing risk to public health, requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here, we report cryo-EM reconstructions of all components of the XM1 virion and describe the atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared to other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection and post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection.