RESUMO
Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce "molecular signposts" that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.
Assuntos
Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , DNA/ultraestrutura , Tomografia com Microscopia Eletrônica , Animais , Aptâmeros de Nucleotídeos/química , Fenômenos Biofísicos , Linhagem Celular , Feminino , Fluorescência , Humanos , Nanopartículas/ultraestruturaRESUMO
Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
Assuntos
Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Vesículas Transportadoras/ultraestrutura , Animais , Capsídeo/ultraestrutura , Chlorocebus aethiops , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Herpesvirus Humano 1/metabolismo , Herpesvirus Suídeo 1/metabolismo , Membrana Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dímeros de Pirimidina , Espalhamento a Baixo Ângulo , Vesículas Transportadoras/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.
Assuntos
Chaperonina 60/química , Trifosfato de Adenosina/metabolismo , Bactérias/química , Bactérias/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Dobramento de ProteínaRESUMO
Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry.
Assuntos
Herpesvirus Humano 1/química , Glicoproteínas de Membrana/química , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica , Conformação ProteicaRESUMO
Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Microtúbulos/ultraestrutura , Citoesqueleto/ultraestrutura , Dineínas/ultraestrutura , Endocitose/fisiologiaRESUMO
Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of â¼20Å, native MLV Env presents as a hollow trimer (height â¼85Å, diameter â¼120Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by â¼35Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition.
Assuntos
Microscopia Crioeletrônica/métodos , Vírus da Leucemia Murina/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Sítios de Ligação , Vírus da Leucemia Murina/fisiologia , Conformação ProteicaRESUMO
UNLABELLED: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE: In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.
Assuntos
Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , Internalização do Vírus , Desenvelopamento do Vírus , Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Microscopia Crioeletrônica , HumanosRESUMO
The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC-capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.
Assuntos
Capsídeo , Núcleo Celular , Microscopia Crioeletrônica , Membrana Nuclear , Liberação de Vírus , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Membrana Nuclear/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Nucleocapsídeo/metabolismo , Tomografia com Microscopia Eletrônica , Proteínas Virais/metabolismo , Proteínas Virais/genética , Herpesviridae/fisiologia , Herpesviridae/genéticaRESUMO
Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV.
Assuntos
Microscopia Crioeletrônica , Vaccinia virus , Vaccinia virus/ultraestrutura , Humanos , Multimerização Proteica , Tomografia com Microscopia Eletrônica , Modelos Moleculares , Vírion/ultraestrutura , Vírion/metabolismoRESUMO
Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen.
Assuntos
Culicidae , Pavilhão Auricular , Parasitos , Humanos , Animais , Microtúbulos , CitoesqueletoRESUMO
Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.
Assuntos
Vírus Gigantes , Mimiviridae , Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Genoma Viral , Vírus Gigantes/genética , Mimiviridae/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Oxirredutases/metabolismoRESUMO
In fitting atomic structures into cryoEM density maps of macromolecular assemblies, the cross-correlation function (CCF) is the most prevalent method of scoring the goodness-of-fit. However, there are still many possible, less studied ways of scoring fits. In this paper, we introduce four scores new to cryoEM fitting and compare their performance to three known scores. Our benchmark consists of (a) 4 protein assemblies with simulated maps at 5-20 Å resolution, including the heptameric ring of GroEL; and (b) 4 experimental maps of GroEL at â¼6-23 Å resolution with corresponding fitted atomic models. We perturb each fit 1000 times and assess each new fit with each score. The correlation between a score and the Cα RMSD of each fit from the "correctly" fitted structure shows that the CCF is one of the best scores, but in certain situations could be augmented or even replaced by other scores. For instance, our implementation of a score based on mutual information outperforms or is comparable to the CCF in almost all test cases, and our new "envelope score" works as well as the CCF at sub-nanometer resolution but is an order of magnitude faster to calculate. The results also suggest that the width of the Gaussian function used to blur the atomic structure into a density map can significantly affect the fitting process. Finally, we show that our score-testing method, when combined with the Laplacian CCF or the mutual information scores, can be used as a statistical tool for improving cryoEM density fitting.
Assuntos
Microscopia Crioeletrônica/métodos , Algoritmos , Chaperonina 60/química , Simulação por Computador , Computação Matemática , Modelos Moleculares , Conformação Proteica , Estatísticas não Paramétricas , Propriedades de SuperfícieRESUMO
MOTIVATION: Single-particle cryo electron microscopy (cryoEM) typically produces density maps of macromolecular assemblies at intermediate to low resolution (approximately 5-30 A). By fitting high-resolution structures of assembly components into these maps, pseudo-atomic models can be obtained. Optimizing the quality-of-fit of all components simultaneously is challenging due to the large search space that makes the exhaustive search over all possible component configurations computationally unfeasible. RESULTS: We developed an efficient mathematical programming algorithm that simultaneously fits all component structures into an assembly density map. The fitting is formulated as a point set matching problem involving several point sets that represent component and assembly densities at a reduced complexity level. In contrast to other point matching algorithms, our algorithm is able to match multiple point sets simultaneously and not only based on their geometrical equivalence, but also based on the similarity of the density in the immediate point neighborhood. In addition, we present an efficient refinement method based on the Iterative Closest Point registration algorithm. The integer quadratic programming method generates an assembly configuration in a few seconds. This efficiency allows the generation of an ensemble of candidate solutions that can be assessed by an independent scoring function. We benchmarked the method using simulated density maps of 11 protein assemblies at 20 A, and an experimental cryoEM map at 23.5 A resolution. Our method was able to generate assembly structures with root-mean-square errors <6.5 A, which have been further reduced to <1.8 A by the local refinement procedure. AVAILABILITY: The program is available upon request as a Matlab code package. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online.
Assuntos
Microscopia Crioeletrônica/métodos , Modelos Teóricos , Algoritmos , Biologia Computacional/métodosRESUMO
Structural determination of molecular complexes by cryo-EM requires large, often complex processing of the image data that are initially obtained. Here, TEMPy2, an update of the TEMPy package to process, optimize and assess cryo-EM maps and the structures fitted to them, is described. New optimization routines, comprehensive automated checks and workflows to perform these tasks are described.
Assuntos
Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Conformação Molecular , Software , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Fluxo de TrabalhoRESUMO
Viruses are obligatory intracellular parasites that reprogram host cells upon infection to produce viral progeny. Here, we review recent structural insights into virus-host interactions in bacteria, archaea, and eukaryotes unveiled by cellular electron cryo-tomography (cryoET). This advanced three-dimensional imaging technique of vitreous samples in near-native state has matured over the past two decades and proven powerful in revealing molecular mechanisms underlying viral replication. Initial studies were restricted to cell peripheries and typically focused on early infection steps, analyzing surface proteins and viral entry. Recent developments including cryo-thinning techniques, phase-plate imaging, and correlative approaches have been instrumental in also targeting rare events inside infected cells. When combined with advances in dedicated image analyses and processing methods, details of virus assembly and egress at (sub)nanometer resolution were uncovered. Altogether, we provide a historical and technical perspective and discuss future directions and impacts of cryoET for integrative structural cell biology analyses of viruses.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Interações entre Hospedeiro e Microrganismos , Imageamento Tridimensional/métodos , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/instrumentação , Replicação Viral , Vírus/ultraestruturaRESUMO
Rabies virus is an important zoonotic pathogen. Its bullet shaped particle contains a helical nucleocapsid. We used cryo-electron tomography and subsequent subtomogram averaging to determine the structure of its ribonucleoprotein. The resulting electron density map allowed for confident fitting of the N-protein crystal structure, indicating that interactions between neighbouring N-proteins are only mediated by N- and C-terminal protruding subdomains (aa 1-27 and aa 355-372). Additional connecting densities, likely stabilizing the ribonucleoprotein complex, are present between neighbouring M-protein densities on the same helical turn and between M- and N-protein densities located on neighbouring helical turns, but not between M-proteins of different turns, as is observed for the related Vesicular stomatitis virus (VSV). This insight into the architecture of the rabies virus nucleocapsid highlights the surprising structural divergence of large biological assemblies even if the building blocks - here exemplified by VSV M- and N-protein - are structurally closely related.
Assuntos
Microscopia Crioeletrônica/métodos , Vírus da Raiva/metabolismo , Vírus da Raiva/ultraestrutura , Raiva/virologia , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Vírion/ultraestrutura , Células HEK293 , Humanos , RNA Viral/análise , RNA Viral/metabolismoRESUMO
We have developed a genetic algorithm for building macromolecular complexes using only a 3D-electron microscopy density map and the atomic structures of the relevant components. For efficient sampling the method uses map feature points calculated by vector quantization. The fitness function combines a mutual information score that quantifies the goodness of fit with a penalty score that helps to avoid clashes between components. Testing the method on ten assemblies (containing 3-8 protein components) and simulated density maps at 10, 15, and 20 Å resolution resulted in identification of the correct topology in 90%, 70%, and 60% of the cases, respectively. We further tested it on four assemblies with experimental maps at 7.2-23.5 Å resolution, showing the ability of the method to identify the correct topology in all cases. We have also demonstrated the importance of the map feature-point quality on assembly fitting in the lack of additional experimental information.
Assuntos
Genótipo , Multimerização Proteica , Software , Algoritmos , Sequência de Aminoácidos , Dados de Sequência Molecular , Dobramento de ProteínaRESUMO
Three-dimensional electron microscopy is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy (template and electron microscopy comparison using Python) is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate-to-low resolution maps, both globally and locally. It also provides procedures for single-fit assessment, ensemble generation of fits, clustering, and multiple and consensus scoring, as well as plots and output files for visualization purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool suitable for both novice and expert structural biologists.
RESUMO
Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.
Assuntos
Transporte Ativo do Núcleo Celular , Herpesviridae/química , Membrana Nuclear/química , Proteínas Nucleares/química , Proteínas Virais/química , Cristalografia por Raios X , Herpesviridae/metabolismo , Modelos Moleculares , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Dedos de ZincoRESUMO
Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins.