Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Feb. 7, 2023

Abstract Conjugation is a major mechanism of horizontal gene transfer promoting the spread antibiotic resistance among human pathogens. It involves establishing junction between donor and recipient cell via an extracellular appendage known as mating pilus. In bacteria, conjugation machinery encoded by plasmids or transposons typically mediates cognate mobile genetic elements. Much less about in archaea. Here, we determine atomic structures cryo-electron microscopy three conjugative pili, two from hyperthermophilic archaea ( Aeropyrum pernix Pyrobaculum calidifontis ) one Ti plasmid bacterium Agrobacterium tumefaciens , show that archaeal pili are homologous to bacterial pili. However, machinery, Ced, has been ‘domesticated’, is, genes for on chromosome rather than elements, cellular DNA.

Language: Английский

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An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Jan. 26, 2024

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

Language: Английский

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Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms

Proceedings of the National Academy of Sciences, Journal Year: 2022, Volume and Issue: 119(26)

Published: June 21, 2022

While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the archaea. It known that extracellular filaments archaea, such as type IV pili, hami, and cannulae, play a part formation of archaeal biofilms. We used cryo-electron microscopy determine atomic structure previously uncharacterized class surface from hyperthermophilic

Language: Английский

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Two distinct archaeal type IV pili structures formed by proteins with identical sequence

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: June 14, 2024

Abstract Type IV pili (T4P) represent one of the most common varieties surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, intercellular communication. Here, we determine atomic structures two distinct adhesive T4P Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both were same polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal where all subunits are equivalent, whereas other tri-pilus, exists three conformations. The conformations tri-pilus very single conformation found involve orientations outer immunoglobulin-like domains, mediated by a flexible linker. Remarkably, domains rotate nearly 180° between mono- Both forms require ATPase TadC-like membrane pore for assembly, indicating that secretion system produce structurally filaments. Our results show appear less constrained rigid than those homologous flagellar filaments as helical propellers.

Language: Английский

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Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles

Cell, Journal Year: 2024, Volume and Issue: 187(20), P. 5604 - 5619.e14

Published: Aug. 28, 2024

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For past 4 years, American insect-rearing facilities have experienced distinctive larval pathology colony collapse farmed Zophobas morio (superworm). By means cryo-EM, we discovered causative agent: densovirus that named black wasting virus (ZmBWV). confirmed etiology disease by fulfilling Koch's postulates characterizing strains from across United States. ZmBWV is member family Parvoviridae with 5,542 nt genome, describe intersubunit interactions explaining its expanded internal volume relative human parvoviruses. Cryo-EM structures at resolutions up 2.1 Å revealed single-strand DNA (ssDNA) ordering capsid inner surface pinned base-binding pockets in surface. Also, demonstrated prophylactic potential non-pathogenic provide cross-protection vivo.

Language: Английский

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Novel Artificial Intelligence-Based Approaches for Ab Initio Structure Determination and Atomic Model Building for Cryo-Electron Microscopy

Micromachines, Journal Year: 2023, Volume and Issue: 14(9), P. 1674 - 1674

Published: Aug. 27, 2023

Single particle cryo-electron microscopy (cryo-EM) has emerged as the prevailing method for near-atomic structure determination, shedding light on important molecular mechanisms of biological macromolecules. However, inherent dynamics and structural variability complexes coupled with large number experimental images generated by a cryo-EM experiment make data processing nontrivial. In particular, ab initio reconstruction atomic model building remain major bottlenecks that demand substantial computational resources manual intervention. Approaches utilizing recent innovations in artificial intelligence (AI) technology, particularly deep learning, have potential to overcome limitations cannot be adequately addressed traditional image approaches. Here, we review newly proposed AI-based methods volume generation, heterogeneous 3D reconstruction, building. We highlight advancements made implementation AI methods, well discuss remaining areas future development.

Language: Английский

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Neck and capsid architecture of the robust Agrobacterium phage Milano

Communications Biology, Journal Year: 2023, Volume and Issue: 6(1)

Published: Sept. 8, 2023

Abstract Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure “neck” region, where DNA-filled capsid is connected to host-recognizing tail remains poorly understood. We describe cryo-EM structures neck, neck-capsid neck-tail junctions, Agrobacterium phage Milano. Milano neck 1 protein connects 12-fold symmetrical a 5-fold vertex icosahedral capsid. Comparison homologs leads four proposed classes, likely evolved from simplest one siphophages more complex ones myo- podophages. surrounded by atypical collar, which covalently crosslinks sheath 1. decorated with three types proteins, minor (mCP) two linking proteins crosslinking mCP major protein. extensive network disulfide bonds within between provides an exceptional structural stability

Language: Английский

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Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry – a perspective

Published: Jan. 16, 2025

Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from secreted proteins in convalescent serum. This aims perform simultaneous sequencing and epitope mapping using combination single particle cryo-electron microscopy (cryoEM) bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate performance deep-learning tool ModelAngelo determining de novo sequences reconstructed 3D volumes antibody-antigen complexes. demonstrate that while map quality is critical bottleneck, it possible sequence variable domains cryoEM reconstructions with accuracies up 80-90%. While rate errors exceeds typical levels somatic hypermutation, show ModelAngelo-derived can be used assign V-genes. provides functional guide assemble peptides LC-MS/MS data more accurately improves tolerance background polyclonal sequences. Following this proof-of-principle, discuss feasibility future directions repertoires.

Language: Английский

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Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry – a perspective

Published: Jan. 16, 2025

Antibodies are a major component of adaptive immunity against invading pathogens. Here we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from secreted proteins in convalescent serum. This aims perform simultaneous sequencing and epitope mapping using combination single particle cryo-electron microscopy (cryoEM) bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate performance deep-learning tool ModelAngelo determining de novo sequences reconstructed 3D volumes antibody-antigen complexes. demonstrate that while map quality is critical bottleneck, it possible sequence variable domains cryoEM reconstructions with accuracies up 80-90%. While rate errors exceeds typical levels somatic hypermutation, show ModelAngelo-derived can be used assign V-genes. provides functional guide assemble peptides LC-MS/MS data more accurately improves tolerance background polyclonal sequences. Following this proof-of-principle, discuss feasibility future directions repertoires.

Language: Английский

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Frontiers in integrative structural modeling of macromolecular assemblies

QRB Discovery, Journal Year: 2025, Volume and Issue: 6

Published: Jan. 1, 2025

Abstract Integrative modeling enables structure determination for large macromolecular assemblies by combining data from multiple experiments with theoretical and computational predictions. Recent advancements in AI-based prediction cryo electron-microscopy have sparked renewed enthusiasm integrative modeling; structures methods can be integrated situ maps to characterize assemblies. This approach previously allowed us others determine the architectures of diverse assemblies, such as nuclear pore complexes, chromatin remodelers, cell–cell junctions. Experimental spanning several scales was used these studies, ranging high-resolution data, X-ray crystallography AlphaFold structure, low-resolution cryo-electron tomography co-immunoprecipitation experiments. Two recurrent challenges emerged across a range studies. First, contained significant fractions disordered regions, necessitating development new regions context ordered regions. Second, needed developed utilize information tomography, timely challenge structural biology is increasingly moving towards characterization. Here, we recapitulate recent developments proteins analysis highlight other opportunities method modeling.

Language: Английский

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