A guided approach for subtomogram averaging of challenging macromolecular assemblies

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.

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 ◽

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.

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 ◽

Radial Spokes ◽

Axonemal Dynein ◽

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.

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 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.01948-06 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4485-4493 ◽

Cited By ~ 61

Author(s):

Claire S. Ting ◽

Chyongere Hsieh ◽

Sesh Sundararaman ◽

Carmen Mannella ◽

Michael Marko

Keyword(s):

Cell Wall ◽

Electron Tomography ◽

Three Dimensional ◽

Membrane System ◽

Niche Differentiation ◽

Dimensional Structure ◽

Comparative Genomic ◽

Photosynthetic Membrane ◽

Peptidoglycan Biosynthesis ◽

Cryo Electron Tomography

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.

The structure of the COPI coat determined within the cell

eLife ◽

10.7554/elife.32493 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 60

Author(s):

Yury S Bykov ◽

Miroslava Schaffer ◽

Svetlana O Dodonova ◽

Sahradha Albert ◽

Jürgen M Plitzko ◽

...

Keyword(s):

Focused Ion Beam ◽

Structural Model ◽

Electron Tomography ◽

Ion Beam ◽

Membrane Thickness ◽

In Vitro Reconstitution ◽

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

Current data processing strategies for cryo-electron tomography and subtomogram averaging

Biochemical Journal ◽

10.1042/bcj20200715 ◽

2021 ◽

Vol 478 (10) ◽

pp. 1827-1845

Author(s):

Euan Pyle ◽

Giulia Zanetti

Keyword(s):

Data Processing ◽

Electron Tomography ◽

Signal To Noise Ratio ◽

Three Dimensional ◽

Current Data ◽

Processing Strategies ◽

Subtomogram Averaging ◽

The Individual

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

A flexible framework for multi-particle refinement in cryo-electron tomography

PLoS Biology ◽

10.1371/journal.pbio.3001319 ◽

2021 ◽

Vol 19 (8) ◽

pp. e3001319

Author(s):

Alister Burt ◽

Lorenzo Gaifas ◽

Tom Dendooven ◽

Irina Gutsche

Keyword(s):

Software Package ◽

Electron Tomography ◽

Macromolecular Structure ◽

Computational Tools ◽

Flexible Framework ◽

Subtomogram Averaging ◽

Particle Refinement ◽

Hiv 1

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

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.

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 ◽

Simplex Virus

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902235116 ◽

2019 ◽

Vol 116 (31) ◽

pp. 15534-15539 ◽

Cited By ~ 6

Author(s):

Thomas Burgoyne ◽

John M. Heumann ◽

Edward P. Morris ◽

Carlo Knupp ◽

Jun Liu ◽

...

Keyword(s):

Actin Filaments ◽

Striated Muscle ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Dimensional Structure ◽

Band Model ◽

Type I ◽

Sonic Muscle ◽

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.