RESUMEN
Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
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The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.
Asunto(s)
Retículo Endoplásmico , Mitocondrias , Saccharomyces cerevisiae , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Lípidos , Mitocondrias/química , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Sinaptotagminas/química , Sinaptotagminas/metabolismoRESUMEN
In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.
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Proteínas de Transporte de Membrana , Ácido N-Acetilneuramínico , Transporte Biológico , Archaea , Adenosina TrifosfatoRESUMEN
We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.
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Electrones , Procesamiento de Imagen Asistido por Computador , Procesamiento de Imagen Asistido por Computador/métodos , Teorema de Bayes , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodosRESUMEN
Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.
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Infecciones por Enterovirus , Poliovirus , Autofagia , Cápside , Fosfatidilinositol 3-Quinasas Clase III , Humanos , Poliovirus/genética , ARN , Virión/genética , Ensamble de Virus/fisiologíaRESUMEN
A semiartificial photosynthesis approach that utilizes enzymes for solar fuel production relies on efficient photosensitizers that should match the enzyme activity and enable long-term stability. Polymer dots (Pdots) are biocompatible photosensitizers that are stable at pH 7 and have a readily modifiable surface morphology. Therefore, Pdots can be considered potential photosensitizers to drive such enzyme-based systems for solar fuel formation. This work introduces and unveils in detail the interaction within the biohybrid assembly composed of binary Pdots and the HydA1 [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The direct attachment of hydrogenase on the surface of toroid-shaped Pdots was confirmed by agarose gel electrophoresis, cryogenic transmission electron microscopy (Cryo-TEM), and cryogenic electron tomography (Cryo-ET). Ultrafast transient spectroscopic techniques were used to characterize photoinduced excitation and dissociation into charges within Pdots. The study reveals that implementation of a donor-acceptor architecture for heterojunction Pdots leads to efficient subpicosecond charge separation and thus enhances hydrogen evolution (88â¯460 µmolH2·gH2ase-1·h-1). Adsorption of [FeFe]-hydrogenase onto Pdots resulted in a stable biohybrid assembly, where hydrogen production persisted for days, reaching a TON of 37â¯500 ± 1290 in the presence of a redox mediator. This work represents an example of a homogeneous biohybrid system combining polymer nanoparticles and an enzyme. Detailed spectroscopic studies provide a mechanistic understanding of light harvesting, charge separation, and transport studied, which is essential for building semiartificial photosynthetic systems with efficiencies beyond natural and artificial systems.
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Chlamydomonas reinhardtii , Hidrogenasas , Proteínas Hierro-Azufre , Hidrógeno/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Fármacos Fotosensibilizantes , PolímerosRESUMEN
Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.
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Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Modelos Moleculares , Programas InformáticosRESUMEN
Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non-Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo-electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.
RESUMEN
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
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Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Beclina-1/química , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Tomografía , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.
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Proteínas de la Cápside/ultraestructura , Infecciones por VIH/genética , VIH-1/genética , Factor de Transcripción Sp1/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/ultraestructura , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/genética , Cristalografía por Rayos X , Infecciones por VIH/virología , Seropositividad para VIH , VIH-1/patogenicidad , VIH-1/ultraestructura , Humanos , Simulación de Dinámica Molecular , Multimerización de Proteína/genética , Proteolisis , Factor de Transcripción Sp1/química , Factor de Transcripción Sp1/genética , Virión/genética , Virión/patogenicidad , Ensamble de Virus/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Synaptic vesicle proteins, including N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Synaptotagmin-1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an in vitro reconstituted fusion system. We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a 'clustered' distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a 'ring-like' arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements.
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Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Membrana Celular/ultraestructura , Microscopía por Crioelectrón , Técnicas In Vitro , Proteínas de la Membrana/ultraestructura , Vesículas Sinápticas/ultraestructura , TomografíaRESUMEN
Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.
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Anemia Infecciosa Equina/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus de la Anemia Infecciosa Equina/fisiología , Ácido Fítico/metabolismo , Virión/fisiología , Secuencia de Aminoácidos , Animales , Tomografía con Microscopio Electrónico , Anemia Infecciosa Equina/virología , Productos del Gen gag/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , VIH-1/ultraestructura , Caballos , Interacciones Huésped-Patógeno , Virus de la Anemia Infecciosa Equina/química , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/ultraestructura , Alineación de Secuencia , Virión/genética , Virión/ultraestructura , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Arc, a neuronal gene that is critical for synaptic plasticity, originated through the domestication of retrotransposon Gag genes and mediates intercellular messenger RNA transfer. We report high-resolution structures of retrovirus-like capsids formed by Drosophila dArc1 and dArc2 that have surface spikes and putative internal RNA-binding domains. These data demonstrate that virus-like capsid-forming properties of Arc are evolutionarily conserved and provide a structural basis for understanding their function in intercellular communication.
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Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestructura , Secuencia de Aminoácidos , Animales , Cápside , Drosophila melanogaster , Conformación ProteicaRESUMEN
The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.
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Proteínas del Citoesqueleto/metabolismo , Exosomas/metabolismo , Productos del Gen gag/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Endocitosis , Femenino , Productos del Gen gag/química , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/fisiologíaRESUMEN
Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane's cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function.
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Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Membrana Celular/química , Membrana Celular/genética , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Membrana Dobles de Lípidos/química , Lípidos/química , Unión ProteicaRESUMEN
The bacterial flagellar motor is a large multi-component molecular machine. Structural determination of such a large complex is often challenging and requires extensive structural analysis in situ. Cryo-electron tomography (cryo-ET) has emerged as a powerful technique that enables us to visualize intact flagellar motors in cells with unprecedented details. Here, we detail the procedure beginning with sample preparation, followed by data acquisition, tomographic reconstruction, sub-tomogram analysis, and ultimately visualization of the intact spirochetal flagellar motor in Borrelia burgdorferi. The procedure is applicable to visualize other molecular machinery in bacteria or other organisms.
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Proteínas Bacterianas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Borrelia burgdorferi/metabolismo , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Flagelos/metabolismoRESUMEN
Bacteriophage SP6 exhibits dual-host adsorption specificity. The SP6 tailspikes are recognized as important in host range determination but the mechanisms underlying dual host specificity are unknown. Cryo-electron tomography and sub-tomogram classification were used to analyze the SP6 virion with a particular focus on the interaction of tailspikes with host membranes. The SP6 tail is surrounded by six V-shaped structures that interconnect in forming a hand-over-hand hexameric garland. Each V-shaped structure consists of two trimeric tailspike proteins: gp46 and gp47, connected through the adaptor protein gp37. SP6 infection of Salmonella enterica serovars Typhimurium and Newport results in distinguishable changes in tailspike orientation, providing the first direct demonstration how tailspikes can confer dual host adsorption specificity. SP6 also infects S. Typhimurium strains lacking O antigen; in these infections tailspikes have no apparent specific role and the phage tail must therefore interact with a distinct host receptor to allow infection.
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Bacteriófagos/fisiología , Salmonella typhimurium/virología , Proteínas de la Cola de los Virus/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Cristalografía por Rayos X , Especificidad del Huésped , Modelos Moleculares , Conformación Proteica , Salmonella typhimurium/clasificación , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/genéticaRESUMEN
Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.
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Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Programas Informáticos , Automatización/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Sustancias Macromoleculares , Shigella flexneri/química , Shigella flexneri/citología , Shigella flexneri/metabolismo , Sistemas de Secreción Tipo III/análisis , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.