scholarly journals Minicells, Back in Fashion

2016 ◽  
Vol 198 (8) ◽  
pp. 1186-1195 ◽  
Author(s):  
Madeline M. Farley ◽  
Bo Hu ◽  
William Margolin ◽  
Jun Liu
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Model Systems ◽  
Specimen Thickness ◽  
Macromolecular Complexes ◽  
Content Type ◽  
Cellular Environment

Cryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational changes in their native cellular environment. However, the resolution and potential applications of cryo-ET are fundamentally limited by specimen thickness, preventing high-resolutionin situvisualization of macromolecular structures in many bacteria (such asEscherichia coliandSalmonella enterica). Minicells, which were discovered nearly 50 years ago, have recently been exploited as model systems to visualize molecular machinesin situ, due to their smaller size and other unique properties. In this review, we discuss strategies for producing minicells and highlight their use in the study of chemotactic signaling, protein secretion, and DNA translocation. In combination with powerful genetic tools and advanced imaging techniques, minicells provide a springboard for in-depth structural studies of bacterial macromolecular complexesin situand therefore offer a unique approach for gaining novel structural insights into many important processes in microbiology.

scholarly journals HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes

2021 ◽  
Vol 8 ◽  
Author(s):  
Mohamad Harastani ◽  
Mikhail Eltsov ◽  
Amélie Leforestier ◽  
Slavica Jonic
Keyword(s):  
Data Analysis ◽  
Rigid Body ◽  
Normal Mode ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Normal Mode Analysis ◽  
Mode Analysis ◽  
Macromolecular Complexes ◽  
Conformational Variability

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

Three-Dimensional Imaging of In Situ Specimens with Low-Dose Electron Tomography to Analyze Protein Conformation

2004 ◽  
Vol 2 (5) ◽  
pp. 561-567 ◽  
Author(s):  
Martina Banyay ◽  
Fredrik Gilstring ◽  
Elenor Hauzenberger ◽  
Lars-Göran Öfverstedt ◽  
Anders B. Eriksson ◽  
...  
Keyword(s):  
Protein Conformation ◽  
Low Dose ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Three Dimensional Imaging

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 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 Proteasomes tether to two distinct sites at the nuclear pore complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13726-13731 ◽  
Author(s):  
Sahradha Albert ◽  
Miroslava Schaffer ◽  
Florian Beck ◽  
Shyamal Mosalaganti ◽  
Shoh Asano ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Cellular Environment ◽  
Subtomogram Averaging ◽  
Substrate Processing ◽  
Cellular Components ◽  
Transport Of Macromolecules

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

Three-dimensional nano-structure of in situ silica in natural rubber as revealed by 3D-TEM/electron tomography

Polymer ◽  
2005 ◽  
Vol 46 (12) ◽  
pp. 4440-4446 ◽  
Author(s):  
Shinzo Kohjiya ◽  
Astushi Katoh ◽  
Junichi Shimanuki ◽  
Toshinori Hasegawa ◽  
Yuko Ikeda
Keyword(s):  
Natural Rubber ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nano Structure ◽  
In Situ Silica

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2019 ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations

AbstractThe nuclear lamina – a meshwork of intermediate filaments termed lamins – functions as a mechanotransduction interface between the extracellular matrix and the nucleus via the cytoskeleton. Although lamins are primarily responsible for the mechanical stability of the nucleus in multicellular organisms, in situ characterization of lamin filaments under tension has remained elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in its three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly under a force of a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough, similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but the meshwork topology follows ‘small world’ properties. Our results suggest that the lamin filaments arrange to form a robust, emergent meshwork that dictates the mechanical properties of individual lamin filaments. The combined approach provides quantitative insights into the structure-function organization of lamins in situ, and implies a role of meshwork topology in laminopathies.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

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