scholarly journals A cryo-ET study of microtubules in axons

2021 ◽  
Author(s):  
Helen E Foster ◽  
Camilla Ventura Santos ◽  
Andrew P Carter
Keyword(s):  
Drosophila Melanogaster ◽  
Dorsal Root ◽  
Cell Shape ◽  
Intracellular Transport ◽  
Electron Tomography ◽  
Structural Features ◽  
Disordered Protein ◽  
Cryo Electron Tomography ◽  
Mouse Dorsal Root Ganglion

The microtubule cytoskeleton in axons plays key roles in intracellular transport and in defining cell shape. Despite many years of study of microtubules, many questions regarding their native architecture remain unanswered. Here, we performed cryo-electron tomography of mouse dorsal root ganglion (DRG) and Drosophila melanogaster (Dm) neurons and examined their microtubule ultrastructure in situ. We found that the microtubule minus and plus ends in DRG axons are structurally similar and frequently contact nearby components. The microtubules in DRG axons maintained a 13 protofilament (pf) architecture, even close to lattice break sites. In contrast, microtubules in Dm neurons had 12 or 13 pfs and we detected sites of pf number transition. The microtubule lumen in DRG axons is filled with globular microtubule inner proteins (MIPs). Our data suggest these have a defined structure, which is surprising given they are thought to contain the disordered protein MAP6. In summary, we reveal novel morphological and structural features of microtubules in their native environment.

scholarly journals DISCA: high-throughput cryo-ET structural pattern mining by deep unsupervised clustering

2021 ◽  
Author(s):  
Xiangrui Zeng ◽  
Anson Kahng ◽  
Liang Xue ◽  
Julia Mahamid ◽  
Yi-Wei Chang ◽  
...  
Keyword(s):  
High Throughput ◽  
Pattern Mining ◽  
Electron Tomography ◽  
Structural Features ◽  
Computer Assisted ◽  
Label Free ◽  
Structural Pattern ◽  
Cryo Electron Tomography ◽  
Homogeneous Structures

Cryo-electron tomography directly visualizes heterogeneous macromolecular structures in complex cellular environments, but existing computer-assisted sorting approaches are low-throughput or inherently limited due to their dependency on available templates and manual labels. We introduce a high-throughput template-and-label-free deep learning approach that automatically discovers subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Diverse structures emerging from sorted subsets enable systematic unbiased recognition of macromolecular complexes in situ.

Cryo electron tomography of vitrified fibroblasts: Microtubule plus ends in situ

2008 ◽  
Vol 161 (3) ◽  
pp. 459-468 ◽  
Author(s):  
Roman I. Koning ◽  
Sandra Zovko ◽  
Montserrat Bárcena ◽  
Gert T. Oostergetel ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography

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 Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

In situ Structure of Viral RNA by Cryo Electron Tomography with Volta Phase Plate, Energy Filtering and Direct Electron Counting

2016 ◽  
Vol 22 (S3) ◽  
pp. 74-75
Author(s):  
Z. Hong Zhou ◽  
Wong H. Hui ◽  
Jiayan Zhang ◽  
Ivo Atanasov ◽  
Cristina C. Celma ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Viral Rna ◽  
Phase Plate ◽  
Energy Filtering ◽  
Electron Counting ◽  
Cryo Electron Tomography

scholarly journals In SituStructures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Shiwei Zhu ◽  
Maren Schniederberend ◽  
Daniel Zhitnitsky ◽  
Ruchi Jain ◽  
Jorge E. Galán ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Electron Tomography ◽  
Additional Insight ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cryo Electron Tomography ◽  
Serovar Typhimurium ◽  
Flagellar Assembly ◽  
Insight Into

ABSTRACTThe bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, includingPseudomonas aeruginosa,Vibriospp., andSalmonella enterica. The bacterial flagellum has been studied extensively in the model systemsEscherichia coliandSalmonella entericaserovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determinein situstructures of polar flagella inP. aeruginosaand peritrichous flagella inS. Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.IMPORTANCEThe bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolutionin situstructures of polar flagella inPseudomonas aeruginosaand peritrichous flagella inSalmonella entericaserovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.

scholarly journals Site Specific Cryo-FIB Preparations Aimed at in situ Cryo-Electron Tomography

2017 ◽  
Vol 23 (S1) ◽  
pp. 250-251
Author(s):  
Julia Mahamid ◽  
Jan Arnold ◽  
Jurgen M. Plitzko
Keyword(s):  
Electron Tomography ◽  
Site Specific ◽  
Cryo Electron Tomography
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