The Dynamo Software Package for Cryo-electron Tomography and Subtomogram Averaging

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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Tools enabling flexible approaches to high-resolution subtomogram averaging

10.1101/2021.01.31.428990 ◽

2021 ◽

Cited By ~ 1

Author(s):

Alister Burt ◽

Lorenzo Gaifas ◽

Tom Dendooven ◽

Irina Gutsche

Keyword(s):

Software Package ◽

State Of The Art ◽

Electron Tomography ◽

Macromolecular Structure ◽

Computational Tools ◽

Cryo Electron Tomography ◽

Subtomogram Averaging ◽

Tomography Data ◽

Hiv 1

AbstractCryo-electron tomography and subtomogram averaging 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 subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

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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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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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Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging

Journal of Structural Biology ◽

10.1016/j.jsb.2016.06.007 ◽

2017 ◽

Vol 197 (2) ◽

pp. 191-198 ◽

Cited By ~ 193

Author(s):

Wim J.H. Hagen ◽

William Wan ◽

John A.G. Briggs

Keyword(s):

High Resolution ◽

Electron Tomography ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

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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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Topological Insights into Mutant Huntingtin Exon 1 and PolyQ Aggregates by Cryo-Electron Tomography

10.1101/2020.12.08.417048 ◽

2020 ◽

Author(s):

Jesús G. Galaz-Montoya ◽

Sarah H. Shahmoradian ◽

Koning Shen ◽

Judith Frydman ◽

Wah Chiu

Keyword(s):

Trinucleotide Repeat ◽

Electron Tomography ◽

Mutant Huntingtin ◽

And Topology ◽

Cryo Electron Tomography ◽

Exon 1 ◽

Subtomogram Averaging ◽

The Brain ◽

Polyq Tract

ABSTRACTHuntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we observed some filaments in both types of aggregates under ∼2 nm in width, thinner than previously reported, while other regions form large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy, slab-shaped morphology in both aggregates, supportive of the “polyQ core” model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling and their identification in cells without fusion tags.

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CryoET structures of immature HIV Gag reveal six-helix bundle

Communications Biology ◽

10.1038/s42003-021-01999-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Luiza Mendonça ◽

Dapeng Sun ◽

Jiying Ning ◽

Jiwei Liu ◽

Abhay Kotecha ◽

...

Keyword(s):

Electron Tomography ◽

Single Amino Acid ◽

Helical Conformation ◽

Particle Assembly ◽

Virus Maturation ◽

Helix Bundle ◽

Cryo Electron Tomography ◽

Subtomogram Averaging ◽

Single Amino Acid Mutation

AbstractGag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

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