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cryo electron tomography

Brianne J Lindsay, Michal M Bonar, Ian N Costas-Cancelas, Kristin Hunt, Alexander I Makarkov, Sabrina Chierzi, Connie M Krawczyk, Nathalie Landry, Brian J Ward, Isabelle Rouiller
Plant-made virus-like particle (VLP) vaccines that display wild-type influenza hemagglutinin (HA) are rapidly advancing through clinical trials. Produced by transient transfection of Nicotiana benthamiana, these novel vaccines are unusually immunogenic, eliciting both humoral and cellular responses. Here, we directly visualized VLPs bearing either HA trimers derived from strains A/California/7/2009 or A/Indonesia/5/05 using cryo-electron microscopy and determined the 3D organization of the VLPs using cryo-electron tomography...
March 14, 2018: Vaccine
Z A Afonina, V A Shirokov
Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed two-row forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes...
January 2018: Biochemistry. Biokhimii︠a︡
Tomohiro Kubo, Yuqing Hou, Deborah A Cochran, George B Witman, Toshiyuki Oda
Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here, we show using cryo-electron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction...
March 14, 2018: Molecular Biology of the Cell
Roman I Koning, Abraham J Koster, Thomas H Sharp
Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware...
March 8, 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
Karen M Davies, Thorsten B Blum, Werner Kühlbrandt
We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated...
March 8, 2018: Proceedings of the National Academy of Sciences of the United States of America
Gang Fu, Qian Wang, Nhan Phan, Paulina Urbanska, Ewa Joachimiak, Jianfeng Lin, Dorota Wloga, Daniela Nicastro
Motile cilia are essential for propelling cells and moving fluids across tissues. The activity of axonemal dynein motors must be precisely coordinated to generate ciliary motility, but their regulatory mechanisms are not well understood. The tether and tether head (T/TH) complex was hypothesized to provide mechanical feedback during ciliary beating because it links the motor domains of the regulatory I1 dynein to the ciliary doublet microtubule. Combining genetic and biochemical approaches with cryo-electron tomography, we identified FAP44 and FAP43 (plus the algae-specific, FAP43-redundant FAP244) as T/TH components...
March 7, 2018: Molecular Biology of the Cell
Danielle A Grotjahn, Saikat Chowdhury, Yiru Xu, Richard J McKenney, Trina A Schroer, Gabriel C Lander
In the version of this article initially published online, an incorrect accession code, EMD-5NW4, was introduced on page 1 of the article PDF, in section 'BICD2N mediates the association of two dynein dimers with a single dynactin'. This has been corrected to PDB 5NW4. The error has been corrected in the PDF and HTML versions of this article.
February 23, 2018: Nature Structural & Molecular Biology
Anja Seybert, Luis Gonzalez-Gonzalez, Margot P Scheffer, Maria Lluch-Senar, Ana M Mariscal, Enrique Querol, Franziska Matthaeus, Jaume Piñol, Achilleas S Frangakis
The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron-dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryo-electron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, in order to systematically analyse the ultrastructural effects...
February 22, 2018: Molecular Microbiology
Michael Elbaum
The three dimensional ultrastructure of cells and tissues comes to light with tomography. There is an inherent tension between representing molecular detail at the highest possible resolution, on one hand, and visualizing spatial relations between large organelles or even neighboring cells in large volumes, on the other. Together with its advantages for pristine sample preservation, cryo-tomography brings particular constraints. A major challenge has been the restriction to specimens thinner than the typical cell...
February 15, 2018: Current Opinion in Microbiology
Muneyoshi Ichikawa, Khanh Huy Bui
Motile eukaryotic cilia and flagella are hair-like organelles responsible for cell motility and mucociliary clearance. Using cryo-electron tomography, it has been shown that the doublet microtubule, the cytoskeleton core of the cilia and flagella, has microtubule inner protein structures binding periodically inside its lumen. More recently, single-particle cryo-electron microscopy analyses of isolated doublet microtubules have shown that microtubule inner proteins form a meshwork inside the doublet microtubule...
February 12, 2018: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
Zhuan Qin, Bo Hu, Jun Liu
Bacterial chemoreceptors form a highly ordered array in concert with the CheA kinase and the CheW coupling protein. The precise architecture of the array is responsible for high sensitivity, high dynamic range, and strong amplification of chemotaxis signaling. Cryo-electron tomography (cryo-ET) has emerged as a unique tool to visualize bacterial chemotaxis arrays at molecular level. Here we describe a detailed cryo-ET and subtomogram averaging procedure to determine in situ structure of the chemoreceptor arrays in Salmonella minicells...
2018: Methods in Molecular Biology
Davide Zabeo, John M Heumann, Cindi L Schwartz, Azusa Suzuki-Shinjo, Garry Morgan, Per O Widlund, Johanna L Höög
Eukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come mostly from studies on protists. Here, cryo-electron tomography (cryo-ET) was performed on intact human spermatozoon tails and showed a variable number of microtubules in the singlet region (inside the end-piece). Inside the microtubule plus end, a novel left-handed interrupted helix which extends several micrometers was discovered...
February 9, 2018: Scientific Reports
Javier Lopez-Garrido, Nikola Ojkic, Kanika Khanna, Felix R Wagner, Elizabeth Villa, Robert G Endres, Kit Pogliano
The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid...
February 8, 2018: Cell
Danielle A Grotjahn, Saikat Chowdhury, Yiru Xu, Richard J McKenney, Trina A Schroer, Gabriel C Lander
Cytoplasmic dynein is a protein complex that transports molecular cargo along microtubules (MTs), playing a key role in the intracellular trafficking network. Vertebrate dynein's movement becomes strikingly enhanced upon interacting with dynactin and a cargo adaptor such as BicaudalD2. However, the mechanisms responsible for increased transport activity are not well understood, largely owing to limited structural information. We used cryo-electron tomography (cryo-ET) to visualize the 3D structure of the MT-bound dynein-dynactin complex from Mus musculus and show that the dynactin-cargo adaptor complex binds two dimeric dyneins...
February 7, 2018: Nature Structural & Molecular Biology
Xinrui Huang, Sha Li, Song Gao
Cryo-electron tomography (cryo-ET) is one of the most advanced technologies for the in situ visualization of molecular machines by producing three-dimensional (3D) biological structures. However, cryo-ET imaging has two serious disadvantages-low dose and low image contrast-which result in high-resolution information being obscured by noise and image quality being degraded, and this causes errors in biological interpretation. The purpose of this research is to explore an optimal wavelet denoising technique to reduce noise in cryo-ET images...
February 7, 2018: Scientific Reports
Van Son Nguyen, Badreddine Douzi, Eric Durand, Alain Roussel, Eric Cascales, Christian Cambillau
The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM...
February 1, 2018: Current Opinion in Structural Biology
Jose-Jesus Fernandez, Sam Li, Tanmay A M Bharat, David A Agard
Recent evidence suggests that the beam-induced motion of the sample during tilt-series acquisition is a major resolution-limiting factor in electron cryo-tomography (cryoET). It causes suboptimal tilt-series alignment and thus deterioration of the reconstruction quality. Here we present a novel approach to tilt-series alignment and tomographic reconstruction that considers the beam-induced sample motion through the tilt-series. It extends the standard fiducial-based alignment approach in cryoET by introducing quadratic polynomials to model the sample motion...
February 2, 2018: Journal of Structural Biology
Kenta Okamoto, Naoyuki Miyazaki, Hemanth K N Reddy, Max F Hantke, Filipe R N C Maia, Daniel S D Larsson, Chantal Abergel, Jean-Michel Claverie, Janos Hajdu, Kazuyoshi Murata, Martin Svenda
Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer...
February 5, 2018: Virology
Qiang Guo, Carina Lehmer, Antonio Martínez-Sánchez, Till Rudack, Florian Beck, Hannelore Hartmann, Manuela Pérez-Berlanga, Frédéric Frottin, Mark S Hipp, F Ulrich Hartl, Dieter Edbauer, Wolfgang Baumeister, Rubén Fernández-Busnadiego
Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia...
January 31, 2018: Cell
Larisa V Kordyukova, Eleonora V Shtykova, Lyudmila A Baratova, Dmitri I Svergun, Oleg V Batishchev
Influenza A virus, a member of the Orthomyxoviridae family of enveloped viruses, is one of the human and animal top killers, and its structure and components are therefore extensively studied during the last decades. The most abundant component, M1 matrix protein, forms a matrix layer (scaffold) under the viral lipid envelope, and the functional roles as well as structural peculiarities of the M1 protein are still under heavy debate. Despite multiple attempts of crystallization, no high resolution structure is available for the full length M1 of Influenza A virus...
February 1, 2018: Journal of Biomolecular Structure & Dynamics
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