Cryo-electron tomography of cells: connecting structure and function

The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (∼1,000 protein subunits, ∼110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo–electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution.

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Viewing Golgi Structure and Function from a Different Perspective—Insights from Electron Tomography

Methods for Analysis of Golgi Complex Function - Methods in Cell Biology ◽

10.1016/b978-0-12-417164-0.00016-1 ◽

2013 ◽

pp. 259-279 ◽

Cited By ~ 6

Author(s):

Brad J. Marsh ◽

Margit Pavelka

Keyword(s):

Electron Tomography ◽

Structure And Function ◽

Golgi Structure ◽

And Function

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Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1367 ◽

2015 ◽

Vol 26 (2) ◽

pp. 294-304 ◽

Cited By ~ 34

Author(s):

Toshiyuki Oda ◽

Haruaki Yanagisawa ◽

Masahide Kikkawa

Keyword(s):

Biochemical Characterization ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Dynein Motor ◽

Cryo Electron Tomography ◽

Regulatory Complex ◽

And Function

The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

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Structural Conservation and Adaptation of the Bacterial Flagella Motor

Biomolecules ◽

10.3390/biom10111492 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1492

Author(s):

Brittany L. Carroll ◽

Jun Liu

Keyword(s):

Electron Tomography ◽

X Ray ◽

Bacterial Flagella ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Cryo Electron Tomography ◽

Flagellar Assembly ◽

Structural Conservation ◽

And Function ◽

Structural Insights

Many bacteria require flagella for the ability to move, survive, and cause infection. The flagellum is a complex nanomachine that has evolved to increase the fitness of each bacterium to diverse environments. Over several decades, molecular, biochemical, and structural insights into the flagella have led to a comprehensive understanding of the structure and function of this fascinating nanomachine. Notably, X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) have elucidated the flagella and their components to unprecedented resolution, gleaning insights into their structural conservation and adaptation. In this review, we focus on recent structural studies that have led to a mechanistic understanding of flagellar assembly, function, and evolution.

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Alternative Architecture of the E. coli Chemosensory Array

Biomolecules ◽

10.3390/biom11040495 ◽

2021 ◽

Vol 11 (4) ◽

pp. 495

Author(s):

Alister Burt ◽

C. Keith Cassidy ◽

Phillip J. Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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Electron Tomography in the Study of Bacterial Structure and Function

Microscopy Today ◽

10.1017/s1551929500050811 ◽

2005 ◽

Vol 13 (1) ◽

pp. 22-25

Author(s):

Kenneth H. Downing ◽

Haixin Sui ◽

Luis R. Comolli ◽

Hoi-Ying Holman

Keyword(s):

Cell Division ◽

Cell Shape ◽

Electron Tomography ◽

Structure And Function ◽

Interacting Proteins ◽

Ultrastructural Studies ◽

Division Site ◽

Metabolic Functions ◽

Bacterial Structure ◽

And Function

Bacteria contain a wealth of mechanisms that organize their internal and external components into a highly polar structure, frequently with distinctive shape, and constrain certain metabolic functions to particular parts of the cell. For example, cell division generally takes place at the middle of the cell, and a host of interacting proteins are involved in ensuring that the division site is positioned properly. Thus, in spite of the lingering perception among some scientists that these cells are simply bags of freely diffusing enzymes, there is much to be learned from ultrastructural studies. Light microscopy has given evidence of cytoskeletal components that presumably establish and maintain cell shape and participate in cell division.

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Alternative Architecture of the E. Coli Chemosensory Array

10.21203/rs.3.rs-124318/v1 ◽

2020 ◽

Author(s):

Alister Burt ◽

C Cassidy ◽

Phillip Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

Abstract Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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Alternative architecture of the E. coli chemosensory array

10.1101/2021.02.05.429929 ◽

2021 ◽

Author(s):

Alister Burt ◽

C. Keith Cassidy ◽

Phillip J. Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

AbstractChemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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