Cryo-Electron Tomography Reveals the Comparative Three-Dimensional Architecture of Prochlorococcus, a Globally Important Marine Cyanobacterium

ABSTRACT In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.

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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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Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

The Journal of Cell Biology ◽

10.1083/jcb.200808050 ◽

2008 ◽

Vol 183 (5) ◽

pp. 923-932 ◽

Cited By ~ 121

Author(s):

Khanh Huy Bui ◽

Hitoshi Sakakibara ◽

Tandis Movassagh ◽

Kazuhiro Oiwa ◽

Takashi Ishikawa

Keyword(s):

Chlamydomonas Reinhardtii ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Molecular Architecture ◽

Heavy Chains ◽

Distal Side ◽

Cryo Electron Tomography ◽

Dynein Arms

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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Faculty Opinions recommendation of Three-dimensional structure of herpes simplex virus from cryo-electron tomography.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1016499.200752 ◽

2003 ◽

Author(s):

Lindsey Hutt-Fletcher

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography ◽

Simplex Virus

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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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Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0692 ◽

2012 ◽

Vol 23 (1) ◽

pp. 111-120 ◽

Cited By ~ 50

Author(s):

Cynthia F. Barber ◽

Thomas Heuser ◽

Blanca I. Carbajal-González ◽

Vladimir V. Botchkarev ◽

Daniela Nicastro

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Flagellar Motility ◽

Three Dimensional Structure ◽

Radial Spoke ◽

Cryo Electron Tomography ◽

Radial Spokes ◽

Axonemal Dynein ◽

Subtomogram Averaging

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.

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Three-Dimensional Structure of Herpes Simplex Virus from Cryo-Electron Tomography

Science ◽

10.1126/science.1090284 ◽

2003 ◽

Vol 302 (5649) ◽

pp. 1396-1398 ◽

Cited By ~ 393

Author(s):

Kay Grünewald ◽

Prashant Desai ◽

Dennis C. Winkler ◽

J. Bernard Heymann ◽

David M. Belnap ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Cryo Electron Tomography ◽

Simplex Virus

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Microtubule architecture in vitro and in cells revealed by cryo-electron tomography

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318001948 ◽

2018 ◽

Vol 74 (6) ◽

pp. 572-584 ◽

Cited By ~ 29

Author(s):

Joseph Atherton ◽

Melissa Stouffer ◽

Fiona Francis ◽

Carolyn A. Moores

Keyword(s):

Molecular Motors ◽

Electron Tomography ◽

Three Dimensional ◽

Structural Heterogeneity ◽

Dimensional Structure ◽

Cellular Functions ◽

Cellular Processes ◽

Cryo Electron Tomography ◽

In Cells

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

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