In Situ Structural Analysis of the Spirochetal Flagellar Motor by Cryo-Electron Tomography

2017 ◽  
pp. 229-242 ◽  
Author(s):  
Shiwei Zhu ◽  
Zhuan Qin ◽  
Juyu Wang ◽  
Dustin R. Morado ◽  
Jun Liu
Keyword(s):  
Structural Analysis ◽  
Electron Tomography ◽  
Flagellar Motor ◽  
Cryo Electron Tomography

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.

scholarly journals Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

2009 ◽  
Vol 191 (16) ◽  
pp. 5026-5036 ◽  
Author(s):  
Jun Liu ◽  
Tao Lin ◽  
Douglas J. Botkin ◽  
Erin McCrum ◽  
Hanspeter Winkler ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Rotational Symmetry ◽  
Flagellar Motor ◽  
Cell Axis ◽  
Ring Assembly ◽  
Flagellar Motors ◽  
Cryo Electron Tomography ◽  
Flagellar Rotation

ABSTRACT The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

Developments in cryo-electron tomography for in situ structural analysis

2015 ◽  
Vol 581 ◽  
pp. 78-85 ◽  
Author(s):  
Anna Dubrovsky ◽  
Simona Sorrentino ◽  
Jan Harapin ◽  
K. Tanuj Sapra ◽  
Ohad Medalia
Keyword(s):  
Structural Analysis ◽  
Electron Tomography ◽  
Cryo Electron Tomography

scholarly journals A lamin A/C variant causing striated muscle disease provides insights into filament organization

2020 ◽  
Author(s):  
Rafael Kronenberg-Tenga ◽  
Meltem Tatli ◽  
Matthias Eibauer ◽  
Wei Wu ◽  
Ji-Yeon Shin ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Striated Muscle ◽  
Electron Tomography ◽  
Nuclear Lamina ◽  
Muscle Disease ◽  
Relative Reduction ◽  
Embryonic Fibroblasts ◽  
Thick Filaments ◽  
Cryo Electron Tomography

AbstractThe LMNA gene encodes the A-type lamins that polymerize into ~3.5 nm thick filaments, and together with B-type lamins and lamin binding proteins form the nuclear lamina. Mutations in LMNA are associated with a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ, and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two-polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

scholarly journals High Throughput Cryo-Electron Tomography: Towards Molecular Architecture of Spirochetal Flagellar Motor in situ

2010 ◽  
Vol 16 (S2) ◽  
pp. 1070-1071
Author(s):  
J Liu ◽  
S Norris
Keyword(s):  
High Throughput ◽  
Electron Tomography ◽  
Flagellar Motor ◽  
Molecular Architecture ◽  
Cryo Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

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.

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