Structural insights into flagellar stator–rotor interactions

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.

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In situstructures of periplasmic flagella inBorreliareveals conformational changes essential for flagellar rotation

10.1101/553289 ◽

2019 ◽

Author(s):

Yunjie Chang ◽

Kihwan Moon ◽

Xiaowei Zhao ◽

J. Norris Steven ◽

Md A. Motaleb ◽

...

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

High Speed ◽

Electron Tomography ◽

Proton Translocation ◽

Three Dimensional ◽

Dimensional Structure ◽

Flagellar Motor ◽

Flagellar Filament ◽

Flagellar Rotation

SUMMARYThe bacterial flagellar motor is a molecular machine that rotates the flagellar filament at high speed. Torque is generated by the stator-rotor interaction coupled to an ion flux through the torque-generating stator. Here, we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirocheteBorrelia burgdorferi. By analysis of the motor structures of wild-type and stator mutants, we localize the torque-generating units precisely and determine three-dimensional structure of the stator and its interactions with the rotor. Our study shows that the cytoplasmic domains of the stator are packed regularly around the circumference of the flagellar C-ring. The stator-rotor interaction induces a profound conformational change in the C-ring. Analysis of the motors of a less motilemotB-D24E mutant and a non-motilemotB-D24N mutant, in which the proton translocation is reduced and blocked, respectively, provides evidence that the conformational change of the C-ring is essential for flagellar rotation.

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

Methods in Molecular Biology - The Bacterial Flagellum ◽

10.1007/978-1-4939-6927-2_18 ◽

2017 ◽

pp. 229-242 ◽

Cited By ~ 7

Author(s):

Shiwei Zhu ◽

Zhuan Qin ◽

Juyu Wang ◽

Dustin R. Morado ◽

Jun Liu

Keyword(s):

Structural Analysis ◽

Electron Tomography ◽

Flagellar Motor ◽

Cryo Electron Tomography

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

Journal of Bacteriology ◽

10.1128/jb.00340-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5026-5036 ◽

Cited By ~ 106

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.

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

Microscopy and Microanalysis ◽

10.1017/s1431927610058836 ◽

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.

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In Situ Structure of the Vibrio Polar Flagellum Reveals a Distinct Outer Membrane Complex and Its Specific Interaction with the Stator

Journal of Bacteriology ◽

10.1128/jb.00592-19 ◽

2019 ◽

Vol 202 (4) ◽

Cited By ~ 5

Author(s):

Shiwei Zhu ◽

Tatsuro Nishikino ◽

Norihiro Takekawa ◽

Hiroyuki Terashima ◽

Seiji Kojima ◽

...

Keyword(s):

High Resolution ◽

Protein Interactions ◽

High Speed ◽

Electron Tomography ◽

Specific Interaction ◽

Protein Protein Interactions ◽

Content Type ◽

Cryo Electron Tomography ◽

Flagellar Rotation

ABSTRACT The bacterial flagellum is a biological nanomachine that rotates to allow bacteria to swim. For flagellar rotation, torque is generated by interactions between a rotor and a stator. The stator, which is composed of MotA and MotB subunit proteins in the membrane, is thought to bind to the peptidoglycan (PG) layer, which anchors the stator around the rotor. Detailed information on the stator and its interactions with the rotor remains unclear. Here, we deployed cryo-electron tomography and genetic analysis to characterize in situ structure of the bacterial flagellar motor in Vibrio alginolyticus, which is best known for its polar sheathed flagellum and high-speed rotation. We determined in situ structure of the motor at unprecedented resolution and revealed the unique protein-protein interactions among Vibrio-specific features, namely the H ring and T ring. Specifically, the H ring is composed of 26 copies of FlgT and FlgO, and the T ring consists of 26 copies of a MotX-MotY heterodimer. We revealed for the first time a specific interaction between the T ring and the stator PomB subunit, providing direct evidence that the stator unit undergoes a large conformational change from a compact form to an extended form. The T ring facilitates the recruitment of the extended stator units for the high-speed motility in Vibrio species. IMPORTANCE The torque of flagellar rotation is generated by interactions between a rotor and a stator; however, detailed structural information is lacking. Here, we utilized cryo-electron tomography and advanced imaging analysis to obtain a high-resolution in situ flagellar basal body structure in Vibrio alginolyticus, which is a Gram-negative marine bacterium. Our high-resolution motor structure not only revealed detailed protein-protein interactions among unique Vibrio-specific features, the T ring and H ring, but also provided the first structural evidence that the T ring interacts directly with the periplasmic domain of the stator. Docking atomic structures of key components into the in situ motor map allowed us to visualize the pseudoatomic architecture of the polar sheathed flagellum in Vibrio spp. and provides novel insight into its assembly and function.

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Faculty Opinions recommendation of Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717047814.792352820 ◽

2012 ◽

Author(s):

Richard Berry

Keyword(s):

Borrelia Burgdorferi ◽

Electron Tomography ◽

Flagellar Motor ◽

Ring Assembly ◽

Cryo Electron Tomography

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

Journal of Structural Biology ◽

10.1016/j.jsb.2007.08.011 ◽

2008 ◽

Vol 161 (3) ◽

pp. 459-468 ◽

Cited By ~ 41

Author(s):

Roman I. Koning ◽

Sandra Zovko ◽

Montserrat Bárcena ◽

Gert T. Oostergetel ◽

Henk K. Koerten ◽

...

Keyword(s):

Electron Tomography ◽

Cryo Electron Tomography

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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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