scholarly journals Recent advances in cryo-electron tomography for in situ structural biology

2019 ◽  
Vol 75 (a2) ◽  
pp. e81-e81
Author(s):  
Juergen Plitzko
Keyword(s):  
Structural Biology ◽  
Electron Tomography ◽  
Recent Advances ◽  
Cryo Electron Tomography

Protocol for cryo-focused ion beam lift-out technique

2019 ◽  
Author(s):  
Miroslava Schaffer ◽  
Stefan Pfeffer ◽  
Julia Mahamid ◽  
Stephan Kleindiek ◽  
Tim Laugks ◽  
...  
Keyword(s):  
High Pressure ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Electron Tomography ◽  
Ion Beam ◽  
Cell Interior ◽  
Cryo Electron Tomography ◽  
Multicellular Organisms

Abstract Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

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 Automated cryo-lamella preparation for high-throughput in-situ structural biology

2019 ◽  
Author(s):  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Cyntia Taveneau ◽  
Hari Venugopal ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Automated Method ◽  
Cellular Environment ◽  
Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

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.

Sign in / Sign up

Export Citation Format

Share Document