Cryogenic electron microscopy structure of human plakophilin-3
Ontology highlight
ABSTRACT:
SUBMITTER: Izard T
PROVIDER: EMPIAR-11567 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description: Not available
| EMPIAR-11029 | biostudies-other
Project description:The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets-all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere.
| S-EPMC4731829 | biostudies-literature
Project description:Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.
| S-EPMC4081473 | biostudies-literature
Project description:Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.
| S-EPMC10754291 | biostudies-literature
Project description:New instrumentation for three-dimensional electron microscopy is facilitating an increase in the throughput of data collection and reconstruction. The increase in throughput creates bottlenecks in the workflow for storing and processing the image data. Here we describe the creation and quantify the throughput of a high-throughput infrastructure supporting collection of three-dimensional data collection.
| S-EPMC3466351 | biostudies-literature
Project description:Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.
| S-EPMC7112279 | biostudies-literature
Project description:Cryogenic transmission electron microscopy (cryo-TEM) is a high-resolution biological imaging method, whereby biological samples, such as purified proteins, macromolecular complexes, viral particles, organelles and cells, are embedded in vitreous ice preserving their native structures. Due to sensitivity of biological materials to the electron beam of the microscope, only relatively low electron doses can be applied during imaging. As a result, the signal arising from the structure of interest is overpowered by noise in the images. To increase the signal-to-noise ratio, different image processing-based strategies that aim at coherent averaging of signal have been devised. In such strategies, images are generally assumed to arise from multiple identical copies of the structure. Prior to averaging, the images must be grouped according to the view of the structure they represent and images representing the same view must be simultaneously aligned relatively to each other. For computational reconstruction of the three-dimensional structure, images must contain different views of the original structure. Structures with multiple symmetry-related substructures are advantageous in averaging approaches because each image provides multiple views of the substructures. However, the symmetry assumption may be valid for only parts of the structure, leading to incoherent averaging of the other parts. Several image processing approaches have been adapted to tackle symmetry-mismatched substructures with increasing success. Such structures are ubiquitous in nature and further computational method development is needed to understanding their biological functions.
| S-EPMC5857907 | biostudies-literature
Project description:Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.
| S-EPMC7457726 | biostudies-literature
Project description:Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built de novo. However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphene-coated grids exhibit a higher b factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.
| S-EPMC10448747 | biostudies-literature
Project description: Not available
| S-EPMC10959069 | biostudies-literature