Three-Dimensional Visualization of the Molecular Architecture of Cell–Cell Junctions In Situ by Cryo-Electron Tomography of Vitreous Sections

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

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A guided approach for subtomogram averaging of challenging macromolecular assemblies

10.1101/2020.02.01.930297 ◽

2020 ◽

Author(s):

Danielle Grotjahn ◽

Saikat Chowdhury ◽

Gabriel C. Lander

Keyword(s):

Electron Tomography ◽

A Priori ◽

Three Dimensional ◽

Structural Heterogeneity ◽

Dimensional Structure ◽

Diverse Range ◽

Macromolecular Assemblies ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

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

Scientific Reports ◽

10.1038/s41598-020-74104-x ◽

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.

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The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin

Molecular Biology of the Cell ◽

10.1091/mbc.e18-05-0331 ◽

2018 ◽

Vol 29 (20) ◽

pp. 2450-2457 ◽

Cited By ~ 28

Author(s):

Shujun Cai ◽

Désirée Böck ◽

Martin Pilhofer ◽

Lu Gan

Keyword(s):

Basic Principle ◽

Electron Tomography ◽

Three Dimensional ◽

Higher Order ◽

Conformational Variability ◽

Repressive Chromatin ◽

Cryo Electron Tomography ◽

Chromatin Folding

The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

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High Throughput Cryo-Electron Tomography: Towards Molecular Architecture of Spirochetal Flagellar Motor in situ

Microscopy and Microanalysis ◽

10.1017/s1431927610058836 ◽

2010 ◽

Vol 16 (S2) ◽

pp. 1070-1071

Author(s):

J Liu ◽

S Norris

Keyword(s):

High Throughput ◽

Electron Tomography ◽

Flagellar Motor ◽

Molecular Architecture ◽

Cryo Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

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In situ architecture of neuronal α-Synuclein inclusions

Nature Communications ◽

10.1038/s41467-021-22108-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Victoria A. Trinkaus ◽

Irene Riera-Tur ◽

Antonio Martínez-Sánchez ◽

Felix J. B. Bäuerlein ◽

Qiang Guo ◽

...

Keyword(s):

Detailed Analysis ◽

Neurodegenerative Disorders ◽

Electron Tomography ◽

Cellular Interaction ◽

Molecular Architecture ◽

Membrane Interactions ◽

Intracellular Membranes ◽

Cryo Electron Tomography ◽

Cellular Organelles

AbstractThe molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution. We show that inclusions seeded by α-Syn aggregates produced recombinantly or purified from patient brain consist of α-Syn fibrils crisscrossing a variety of cellular organelles. Using gold-labeled seeds, we find that aggregate seeding is predominantly mediated by small α-Syn fibrils, from which cytoplasmic fibrils grow unidirectionally. Detailed analysis of membrane interactions revealed that α-Syn fibrils do not contact membranes directly, and that α-Syn does not drive membrane clustering. Altogether, we conclusively demonstrate that neuronal α-Syn inclusions consist of α-Syn fibrils intermixed with membranous organelles, and illuminate the mechanism of aggregate seeding and cellular interaction.

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Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

10.1101/2021.12.31.474669 ◽

2022 ◽

Author(s):

Jie E Yang ◽

Matthew R Larson ◽

Bryan S Sibert ◽

Joseph Y Kim ◽

Daniel Parrell ◽

...

Keyword(s):

Focused Ion Beam ◽

Low Dose ◽

Structural Information ◽

Electron Tomography ◽

Ion Beam ◽

Three Dimensional ◽

Structural Studies ◽

Cryo Electron Tomography ◽

Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

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