RESUMO
Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.
Assuntos
Microscopia Crioeletrônica , Vírus Sincicial Respiratório Humano , Proteínas Virais de Fusão , Proteínas da Matriz Viral , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/ultraestrutura , Humanos , Vírus Sincicial Respiratório Humano/química , Multimerização Proteica , Vírion/metabolismo , Vírion/ultraestrutura , Vírion/química , Tomografia com Microscopia Eletrônica , Vírus Sinciciais Respiratórios/química , Modelos Moleculares , Infecções por Vírus Respiratório Sincicial/virologia , AnimaisRESUMO
Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP1,2) are the standard of care for Ebola virus disease (EVD). Anti-GP1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro. However, their neutralization mechanism is poorly understood because they target a GP1,2 epitope that has evaded structural characterization. Moreover, their in vivo efficacy has only been evaluated in the mouse model of EVD. Using x-ray crystallography and cryo-electron tomography of 3A6 complexed with its stalk- GP1,2 MPER epitope we reveal a novel mechanism in which 3A6 elevates the stalk or stabilizes a conformation of GP1,2 that is lifted from the virion membrane. In domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high viremia levels, advanced disease stages, and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. These findings can guide design of next-generation, highly potent anti-EBOV mAbs.
RESUMO
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.
Assuntos
Elétrons , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Teorema de Bayes , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Algoritmos , Enzima de Conversão de Angiotensina 2/química , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores Virais/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Ligação Viral , Internalização do VírusRESUMO
Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.
Assuntos
Antígenos HIV/química , Antígenos HIV/metabolismo , HIV-1/química , HIV-1/fisiologia , Envelope Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Capsídeo/química , Capsídeo/fisiologia , Tomografia com Microscopia Eletrônica , HIV-1/ultraestrutura , Bicamadas Lipídicas , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Envelope Viral/química , Envelope Viral/ultraestrutura , Vírion/química , Vírion/fisiologia , Vírion/ultraestrutura , Montagem de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind, fuse, and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes the preparation of the fusion machinery by dissociation of the S1 domains. We report the development of bottom-up coarse-grained (CG) models validated with cryo-electron tomography (cryo-ET) data, and the use of CG molecular dynamics simulations to investigate the dynamical mechanisms involved in viral binding and exposure of the S2 trimeric core. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces. The multivalent interaction cyclically and processively induces S1 dissociation, thereby exposing the S2 core containing the fusion machinery. Our simulations thus reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.
RESUMO
HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.
RESUMO
Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ÏCbK and ÏCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ÏCbK adsorption efficiency, although no single deletion completely ablates ÏCbK adsorption. Thus, the bacteriophage ÏCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ÏCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ÏCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ÏCbK infects C. crescentus at the molecular level.IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ÏCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ÏCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ÏCbK attaches to flagellar filaments at the molecular level.
Assuntos
Bacteriófagos/fisiologia , Caulobacter crescentus/ultraestrutura , Caulobacter crescentus/virologia , Flagelos/química , Flagelina/química , Ligação Viral , Sequência de Aminoácidos , Caulobacter crescentus/genética , Flagelina/genética , Genes Bacterianos , Conformação Proteica em alfa-HéliceRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
Assuntos
Microscopia Crioeletrônica , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Vírion/química , Vírion/ultraestrutura , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Maleabilidade , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Vírion/isolamento & purificação , Vírion/metabolismoRESUMO
The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. It exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor-binding site and, subsequently, from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S-protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S that allow the production of thermostable, disulfide-bonded S-protein trimers that are trapped in the closed, prefusion state. Structures of the disulfide-stabilized and non-disulfide-stabilized proteins reveal distinct closed and locked conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.
Assuntos
Betacoronavirus/química , Betacoronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Betacoronavirus/genética , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Microscopia Crioeletrônica , Dissulfetos/química , Humanos , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Engenharia de Proteínas/métodos , Multimerização Proteica , Estabilidade Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , TemperaturaRESUMO
Human hepatitis B virus core protein (HBc) is a structural protein of the hepatitis B virus (HBV) and contributes to HBV regulation of host-cell transcription. However, the mechanisms of transcriptional regulation remain poorly characterized. To dissect the function of HBc, a yeast two-hybrid was performed to identify HBc-binding proteins, and the C-terminal of BRG1/hBRM-associated factors 200 (BAF200C) was identified. Then, the existence of HBc interactions with BAF200C and full-length BAF200 was confirmed via co-immunoprecipitation assays in 293T, HepG2 and HepG2-NTCP cells. Furthermore, we show that the binding between HBc and BAF200 was of vital importance to HBc mediated downregulation of interferon-induced transmembrane protein 1 (IFITM1) expression, and the mechanisms for the downregulation were disclosed as follows. Basal level of IFITM1 expression depends on BAF200, rather than the JAK-STAT1 pathway. The interaction of HBc with BAF200 disturbs the stability of the polybromo-associated BAF (PBAF) complex and results in the suppression of IFTM1 transcription. Finally, the antiviral effects of IFITM1 on cell proliferation and HBV replication were found to be partially restored when HBc was co-transfected with BAF200. Collectively, our findings indicate that HBc plays a role in HBV resistance against the antiviral activities of IFNα, providing details about HBV evasion of host innate immunity.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Interferon-alfa/metabolismo , Fatores de Transcrição/metabolismo , Antígenos de Diferenciação/genética , Células Hep G2 , Hepatite B/genética , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/análise , Manejo de Espécimes/métodosRESUMO
Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.
Assuntos
Vírus Sincicial Respiratório Humano/ultraestrutura , Vírion/ultraestrutura , Montagem de Vírus/fisiologia , Células A549 , Animais , Brônquios/virologia , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Células HeLa , Humanos , Microtomia , Vírus Sincicial Respiratório Humano/fisiologia , Células Vero , Vírion/fisiologiaRESUMO
Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.
Assuntos
Hemaglutininas Virais/ultraestrutura , Vírus do Sarampo/ultraestrutura , Ribonucleoproteínas/ultraestrutura , Proteínas Virais de Fusão/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura , Vírion/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Células HeLa , Hemaglutininas Virais/metabolismo , Humanos , Vírus do Sarampo/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Liberação de VírusRESUMO
It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized Caulobacter crescentus pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.
Assuntos
Caulobacter crescentus/fisiologia , Fímbrias Bacterianas/fisiologia , Aderência Bacteriana , Caulobacter crescentus/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismoRESUMO
Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas da Mielina/metabolismo , Membrana Celular/ultraestrutura , Doença de Charcot-Marie-Tooth , Cisteína/química , Cisteína/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Lipossomos/química , Lipossomos/ultraestrutura , Mutação , Proteínas da Mielina/química , Proteínas da Mielina/genética , Proteínas RecombinantesRESUMO
Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.
Assuntos
Elastina/química , Peptídeos/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Elastina/ultraestrutura , Proteínas de Membrana/química , Nefelometria e Turbidimetria , Difração de Nêutrons , Transição de Fase , Proteínas Recombinantes de Fusão/ultraestrutura , Espalhamento a Baixo Ângulo , Tensoativos/química , TemperaturaRESUMO
Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.