RESUMEN
Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20× the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.
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Tomografía con Microscopio Electrónico , Plata , InvestigaciónRESUMEN
INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].
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Inteligencia Artificial , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Proteínas de Xenopus , Animales , Microscopía por Crioelectrón , Citosol/metabolismo , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Oocitos , Conformación Proteica , Subunidades de Proteína/metabolismo , Programas Informáticos , Proteínas de Xenopus/química , Xenopus laevis/metabolismoRESUMEN
Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.
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Tomografía con Microscopio Electrónico , Electrones , Microscopía por Crioelectrón , Congelación , Humanos , Microscopía Electrónica de RastreoRESUMEN
Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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Microscopía por Crioelectrón/métodos , Orthoreovirus/ultraestructura , Animales , Cápside/ultraestructura , Línea Celular , Tomografía con Microscopio Electrónico/métodos , Virión/ultraestructura , Ensamble de VirusRESUMEN
Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.
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Estructuras Citoplasmáticas/ultraestructura , Imagenología Tridimensional/métodos , Saccharomyces cerevisiae/ultraestructura , Biomarcadores/metabolismo , Microscopía por Crioelectrón , Estructuras Citoplasmáticas/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Imagenología Tridimensional/instrumentación , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , VitrificaciónRESUMEN
Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.
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Proteínas de la Cápside/inmunología , Quimera/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Cápside/inmunología , Línea Celular , Quimera/genética , Cricetinae , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Cabras , PorcinosRESUMEN
We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.
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Cristalografía por Rayos X/métodos , Microtecnología/métodos , Muramidasa/ultraestructura , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/instrumentación , Electrones , Iones , Microtecnología/instrumentación , Muramidasa/química , SincrotronesRESUMEN
Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a "ground truth" for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.
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Tomografía con Microscopio Electrónico/métodos , Tomografía con Microscopio Electrónico/normas , Algoritmos , Reproducibilidad de los ResultadosRESUMEN
Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.
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Algoritmos , Tomografía con Microscopio Electrónico/métodos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/normas , Virión/ultraestructuraRESUMEN
Electron cryo-microscopy has become a routine technique to determine the structure of biochemically purified herpes simplex virus capsid particles. This chapter describes the procedures of specimen preparation by cryopreservation; low dose and low temperature imaging in an electron cryo-microscope; and data processing for reconstruction. This methodology has yielded subnanometer resolution structures of the icosahedral capsid shell where α-helices and ß-sheets of individual subunits can be recognized. A relaxation of the symmetry in the reconstruction steps allows us to resolve the DNA packaging protein located at one of the 12 vertices in the capsid.
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Proteínas de la Cápside/ultraestructura , Cápside/ultraestructura , Herpesvirus Humano 1/genética , Biología Molecular/métodos , Proteínas de la Cápside/química , Microscopía por Crioelectrón , Empaquetamiento del ADN/genética , Herpesvirus Humano 1/química , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.
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MicroARNs/genética , Infecciones por Polyomavirus/virología , ARN Viral/genética , Virus 40 de los Simios/genética , Infecciones Tumorales por Virus/virología , Animales , Cricetinae , Femenino , Masculino , Mesocricetus , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/patología , Carga ViralRESUMEN
Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture.