Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution 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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The Dynamo Software Package for Cryo-electron Tomography and Subtomogram Averaging

Microscopy and Microanalysis ◽

10.1017/s1431927620023958 ◽

2020 ◽

Vol 26 (S2) ◽

pp. 3142-3145

Author(s):

Paula Navarro ◽

Stefano Scaramuzza ◽

Henning Stahlberg ◽

Daniel Castaño-Díez

Keyword(s):

Software Package ◽

Electron Tomography ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

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A mammalian system for high-resolution imaging of intact cells by cryo-electron tomography

Progress in Biophysics and Molecular Biology ◽

10.1016/j.pbiomolbio.2020.09.005 ◽

2020 ◽

Author(s):

Xia Li ◽

Donghyun Park ◽

Yunjie Chang ◽

Abhijith Radhakrishnan ◽

Hangjun Wu ◽

...

Keyword(s):

High Resolution ◽

Electron Tomography ◽

High Resolution Imaging ◽

Intact Cells ◽

Cryo Electron Tomography ◽

Resolution Imaging ◽

Mammalian System

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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 ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

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.

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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 ◽

Cryo Electron Tomography ◽

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.

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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 ◽

Cryo Electron Tomography ◽

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.

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