scholarly journals Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian K. M. Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Active Conformation ◽  
Structural Rearrangements ◽  
Cryo Electron Tomography ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.

scholarly journals Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian KM Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Branch Formation ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

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.

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

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

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

scholarly journals Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

Structure ◽  
2016 ◽  
Vol 24 (7) ◽  
pp. 1031-1043 ◽  
Author(s):  
Marion Jasnin ◽  
Mary Ecke ◽  
Wolfgang Baumeister ◽  
Günther Gerisch
Keyword(s):  
Electron Tomography ◽  
Live Imaging ◽  
Actin Organization ◽  
Cryo Electron Tomography ◽  
In Cells

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

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.

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

PLoS Biology ◽  
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.

scholarly journals The dynamic instability of actin filament barbed ends

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Guillaume Romet-Lemonne ◽  
Antoine Jégou
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Dynamic Instability ◽  
Filament Networks ◽  
In Cells

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

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

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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