scholarly journals In situ structure determination of virus capsids imaged within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

AbstractCryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures at atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (>500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised equipment of limited availability. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions reveal that the capsid associated tegument complex is present on capsids prior to nuclear egress. We show that this approach to cryogenic imaging of cells is suited to both correlative light/electron microscopy and 3D structure determination.

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

scholarly journals 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography

2012 ◽  
Vol 180 (2) ◽  
pp. 318-326 ◽  
Author(s):  
Ke Wang ◽  
Korrinn Strunk ◽  
Gongpu Zhao ◽  
Jennifer L. Gray ◽  
Peijun Zhang
Keyword(s):  
Structure Determination ◽  
Mammalian Cells ◽  
Electron Tomography ◽  
3D Structure ◽  
Cryo Electron Tomography ◽  
3D Structure Determination

scholarly journals User Manual for Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS)

2019 ◽  
Author(s):  
Zhiguo Shang ◽  
Kangkang Song ◽  
Xiaofeng Fu ◽  
Xiaochu Lou ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Intact Cells ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Crowded Environment

Abstract Recent advances in cryo-electron microscopy (cryo-EM) are paving the way to determining isolated three-dimensional (3D) macromolecular structures at near-atomic resolution using single-particle cryo-electron microscopy (SP-cryo-EM). However, determining the subcellular structures in intact cells and organelles using cryo-electron tomography (cryo-ET) and subtomogram averaging, another cryo-EM technique, with comparable resolution remains a challenge. Current methodologies can only reach a resolution of several nanometers in most samples studied. Here, we introduce a new hybrid method, called Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS) that is able to achieve structural determination of subcellular structures within their natural crowded environment with nanometer-resolution by combining the advantages of cryo-ET and SP-cryo-EM.

scholarly journals Electron Tomography: Quantitative Structure Determination of Large Three-Dimensional Nanoparticle Assemblies (Part. Part. Syst. Charact. 1/2013)

2012 ◽  
Vol 30 (1) ◽  
pp. 83-83
Author(s):  
Thomas Altantzis ◽  
Bart Goris ◽  
Ana Sánchez-Iglesias ◽  
Marek Grzelczak ◽  
Luis M. Liz-Marzán ◽  
...  
Keyword(s):  
Structure Determination ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Quantitative Structure ◽  
Nanoparticle Assemblies

scholarly journals Nanoscale characterization of the biomolecular corona by cryo-electron microscopy, cryo-electron tomography, and image simulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Sheibani ◽  
Kaustuv Basu ◽  
Ali Farnudi ◽  
Aliakbar Ashkarran ◽  
Muneyoshi Ichikawa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Three Dimensional ◽  
True Nature ◽  
Cryo Electron Microscopy ◽  
Wide Range ◽  
Cryo Electron Tomography ◽  
Nanoscale Characterization ◽  
Dimensional Reconstruction

AbstractThe biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.

scholarly journals Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

2019 ◽  
Author(s):  
Gang Fu ◽  
Lei Zhao ◽  
Erin Dymek ◽  
Yuqing Hou ◽  
Kangkang Song ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Protein Composition ◽  
Wild Type ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Central Apparatus ◽  
Subunit Organization

AbstractNearly all motile cilia contain a central apparatus (CA) composed of two connected singlet-microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild type Chlamydomonas with CA mutants. We identified a large (>2 MDa) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold-labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and three-dimensional (3D) structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.SummaryFu et al. use a wild-type vs. mutant comparison and cryo-electron tomography of Chlamydomonas flagella to identify central apparatus (CA) subunits and visualize their location in the native 3D CA structure. The study provides a better understanding of the CA and how it regulates ciliary motility.

scholarly journals The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 446 ◽  
Author(s):  
Zunlong Ke ◽  
Rebecca Dillard ◽  
Tatiana Chirkova ◽  
Fredrick Leon ◽  
Christopher Stobart ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Electron Tomography ◽  
Rna Virus ◽  
3D Structure ◽  
Three Dimensional ◽  
The Elderly ◽  
Cryo Electron Tomography ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Tract Disease

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.

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