scholarly journals Visualizing the Molecular Machines of Bacterial Motility In-situ with Electron Cryotomography

2006 ◽  
Vol 12 (S02) ◽  
pp. 398-399
Author(s):  
GJ Jensen ◽  
GE Murphy ◽  
GP Henderson ◽  
Z Li ◽  
A Komeili ◽  
...  
Keyword(s):  
Molecular Machines ◽  
Bacterial Motility ◽  
Electron Cryotomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

scholarly journals In situ structure and organization of the influenza C virus surface glycoprotein

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Steinar Halldorsson ◽  
Kasim Sader ◽  
Jack Turner ◽  
Lesley J. Calder ◽  
Peter B. Rosenthal
Keyword(s):  
Membrane Fusion ◽  
Hexagonal Lattice ◽  
Surface Glycoprotein ◽  
Structural Basis ◽  
Virus Surface ◽  
The Matrix ◽  
Influenza C Virus ◽  
Subtomogram Averaging ◽  
Electron Cryotomography

AbstractThe lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF’s membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

scholarly journals Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

2016 ◽  
Vol 113 (30) ◽  
pp. 8442-8447 ◽  
Author(s):  
Alexander W. Mühleip ◽  
Friederike Joos ◽  
Christoph Wigge ◽  
Achilleas S. Frangakis ◽  
Werner Kühlbrandt ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Protein Complexes ◽  
Paramecium Tetraurelia ◽  
Mitochondrial Atp Synthase ◽  
Structural Differences ◽  
Membrane Protein Complexes ◽  
Subtomogram Averaging ◽  
Electron Cryotomography ◽  
Energy Converting

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

scholarly journals Structure and function of a bacterial gap junction analog

2018 ◽  
Author(s):  
Gregor L. Weiss ◽  
Ann-Katrin Kieninger ◽  
Iris Maldener ◽  
Karl Forchhammer ◽  
Martin Pilhofer
Keyword(s):  
Intercellular Communication ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Junction ◽  
Domains Of Life ◽  
A Cell ◽  
Electron Cryotomography ◽  
And Function ◽  
Sister Cells

SummaryMulticellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood and it is unknown whether they regulate intercellular communication.Here we resolved thein situarchitecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacteria. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a plug that was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. This gating was accompanied by a conformational change of the septal junctions, mediated by the proteins FraC/D.We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange, e.g. upon injury.

scholarly journals A collection of yeast cellular electron cryotomography data

GigaScience ◽  
2019 ◽  
Vol 8 (6) ◽  
Author(s):  
Lu Gan ◽  
Cai Tong Ng ◽  
Chen Chen ◽  
Shujun Cai
Keyword(s):  
Cell Biology ◽  
Template Matching ◽  
Feature Recognition ◽  
Yeast Cells ◽  
Large Set ◽  
Macromolecular Complexes ◽  
Structural Cell ◽  
Tilt Series ◽  
Electron Cryotomography

Abstract Background Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms—3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. Findings Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. Conclusions Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.

Structure of the Legionella Dot/Icm type IV secretion system in situ by electron cryotomography

2016 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwangcheol C. Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Structural Preservation ◽  
Electron Cryotomography ◽  
Type Ivb Secretion System

AbstractType IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence homology with type IVA secretion systems (T4ASSs), its overall structure shows remarkable similarities to two previously imaged T4ASSs, suggesting shared aspects of mechanism. However, compared to one of these, the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, it is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

scholarly journals Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

2018 ◽  
Vol 218 (2) ◽  
pp. 455-473 ◽  
Author(s):  
Cai Tong Ng ◽  
Li Deng ◽  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Depolymerization ◽  
Yeast Chromosome ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Electron Cryotomography ◽  
Binding Position

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

scholarly journals Insights into the evolution of bacterial flagellar motors from high-throughput in situ electron cryotomography and subtomogram averaging

2018 ◽  
Vol 74 (6) ◽  
pp. 585-594 ◽  
Author(s):  
Florian M. Rossmann ◽  
Morgan Beeby
Keyword(s):  
High Throughput ◽  
Structural Information ◽  
Bacterial Species ◽  
Phylogenetic Reconstruction ◽  
Structural Diversity ◽  
Phenotypic Analysis ◽  
Assembly Mechanism ◽  
Flagellar Motors ◽  
Electron Cryotomography

In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions.

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