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cryo electron tomography

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https://www.readbyqxmd.com/read/29748979/quantitative-cryo-scanning-transmission-electron-microscopy-of-biological-materials
#1
REVIEW
Michael Elbaum
Electron tomography provides a detailed view into the 3D structure of biological cells and tissues. Physical fixation by vitrification of the aqueous medium provides the most faithful preservation of biological specimens in the native, fully hydrated state. Cryo-microscopy is challenging, however, because of the sensitivity to electron irradiation and due to the weak electron scattering of organic material. Tomography is even more challenging because of the dependence on multiple exposures of the same area...
May 11, 2018: Advanced Materials
https://www.readbyqxmd.com/read/29741156/systematic-studies-of-all-pih-proteins-in-zebrafish-reveal-their-distinct-roles-in-axonemal-dynein-assembly
#2
Hiroshi Yamaguchi, Toshiyuki Oda, Masahide Kikkawa, Hiroyuki Takeda
Construction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1 , pih1d2 , ktu , and twister , and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography...
May 9, 2018: ELife
https://www.readbyqxmd.com/read/29727463/chlamydia-exploits-filopodial-capture-and-a-macropinocytosis-like-pathway-for-host-cell-entry
#3
Charlotte Ford, Andrea Nans, Emmanuel Boucrot, Richard D Hayward
Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway...
May 4, 2018: PLoS Pathogens
https://www.readbyqxmd.com/read/29712906/promotion-of-virus-assembly-and-organization-by-the-measles-virus-matrix-protein
#4
Zunlong Ke, Joshua D Strauss, Cheri M Hampton, Melinda A Brindley, Rebecca S Dillard, Fredrick Leon, Kristen M Lamb, Richard K Plemper, Elizabeth R Wright
Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles...
April 30, 2018: Nature Communications
https://www.readbyqxmd.com/read/29700238/asymmetric-distribution-and-spatial-switching-of-dynein-activity-generates-ciliary-motility
#5
Jianfeng Lin, Daniela Nicastro
Motile cilia and flagella are essential, highly conserved organelles, and their motility is driven by the coordinated activities of multiple dynein isoforms. The prevailing "switch-point" hypothesis posits that dyneins are asymmetrically activated to drive flagellar bending. To test this model, we applied cryo-electron tomography to visualize activity states of individual dyneins relative to their locations along beating flagella of sea urchin sperm cells. As predicted, bending was generated by the asymmetric distribution of dynein activity on opposite sides of the flagellum...
April 27, 2018: Science
https://www.readbyqxmd.com/read/29681471/correlative-microscopy-of-vitreous-sections-provides-insights-into-bar-domain-organization-in-situ
#6
Tanmay A M Bharat, Patrick C Hoffmann, Wanda Kukulski
Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes...
April 10, 2018: Structure
https://www.readbyqxmd.com/read/29678879/potassium-is-a-trigger-for-conformational-change-in-the-fusion-spike-of-an-enveloped-rna-virus
#7
Emma K Punch, Samantha Hover, Henry Tw Blest, Jack Fuller, Roger Hewson, Juan Fontana, Jamel Mankouri, John N Barr
Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the changing endosomal environment, which triggers conformational change in the spike that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition and endosome-resident proteins and these cues dictate when and where viral fusion occurs...
April 20, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29677208/modeling-the-dynamics-and-kinetics-of-hiv-1-gag-during-viral-assembly
#8
Michael D Tomasini, Daniel S Johnson, Joshua S Mincer, Sanford M Simon
We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions...
2018: PloS One
https://www.readbyqxmd.com/read/29666871/harmonic-analysis-of-surface-instability-patterns-on-colloidal-particles
#9
Tero Kämäräinen, Mariko Ago, Jani Seitsonen, Janne Raula, Esko I Kauppinen, Janne Ruokolainen, Orlando J Rojas
Wrinkling of colloidal particles alter a wide variety of interfacial properties but quantitative topographical descriptions have been explored experimentally to a very limited extent. In this study, we present a harmonic analysis of surface wrinkles and folds on submicron colloidal particles, obtained using an aerosol flow route, with small radius (<300 nm) and high crust thickness-to-radius ratio (>0.1). The particle surface coordinates were mapped in their entirety using cryo-electron tomography and subsequently reconstructed using spherical harmonics, allowing a spectral topographical description of the instability patterns and the identification of their surface modes by lateral wavelength...
April 18, 2018: Soft Matter
https://www.readbyqxmd.com/read/29656089/microbiology-catches-the-cryo-em-bug
#10
REVIEW
Lesley A Earl, Veronica Falconieri, Sriram Subramaniam
Over the past few years, the advances in technology and methods that have revolutionized cryo-EM are allowing for key insights in a variety of areas in biology, and microbiology is no exception. A wide range of important macromolecular assemblies in prokaryotic and eukaryotic cells, as well as intact viruses, have now become accessible to investigation by new methods in 3D electron microscopy. We focus here on selected examples that illustrate this breadth, and review the application of methods in single particle cryo-EM and cryo-electron tomography to progress in the structural biology of CRISPR systems, visualization of small molecule drugs in membrane proteins, in situ visualization of bacterial nanomachines, and the analysis of antigen-antibody interactions to drive vaccine design...
April 12, 2018: Current Opinion in Microbiology
https://www.readbyqxmd.com/read/29605906/high-throughput-electron-cryo-tomography-of-protein-complexes-and-their-assembly
#11
Louie D Henderson, Morgan Beeby
Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29581260/molecular-and-structural-architecture-of-polyq-aggregates-in-yeast
#12
Anselm Gruber, Daniel Hornburg, Matthias Antonin, Natalie Krahmer, Javier Collado, Miroslava Schaffer, Greta Zubaite, Christian Lüchtenborg, Timo Sachsenheimer, Britta Brügger, Matthias Mann, Wolfgang Baumeister, F Ulrich Hartl, Mark S Hipp, Rubén Fernández-Busnadiego
Huntington's disease is caused by the expansion of a polyglutamine (polyQ) tract in the N-terminal exon of huntingtin (HttEx1), but the cellular mechanisms leading to neurodegeneration remain poorly understood. Here we present in situ structural studies by cryo-electron tomography of an established yeast model system of polyQ toxicity. We find that expression of polyQ-expanded HttEx1 results in the formation of unstructured inclusion bodies and in some cases fibrillar aggregates. This contrasts with recent findings in mammalian cells, where polyQ inclusions were exclusively fibrillar...
March 26, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29570700/fluctuating-finite-element-analysis-ffea-a-continuum-mechanics-software-tool-for-mesoscale-simulation-of-biomolecules
#13
Albert Solernou, Benjamin S Hanson, Robin A Richardson, Robert Welch, Daniel J Read, Oliver G Harlen, Sarah A Harris
Fluctuating Finite Element Analysis (FFEA) is a software package designed to perform continuum mechanics simulations of proteins and other globular macromolecules. It combines conventional finite element methods with stochastic thermal noise, and is appropriate for simulations of large proteins and protein complexes at the mesoscale (length-scales in the range of 5 nm to 1 μm), where there is currently a paucity of modelling tools. It requires 3D volumetric information as input, which can be low resolution structural information such as cryo-electron tomography (cryo-ET) maps or much higher resolution atomistic co-ordinates from which volumetric information can be extracted...
March 23, 2018: PLoS Computational Biology
https://www.readbyqxmd.com/read/29550194/morphological-characterization-of-a-plant-made-virus-like-particle-vaccine-bearing-influenza-virus-hemagglutinins-by-electron-microscopy
#14
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
https://www.readbyqxmd.com/read/29544430/three-dimensional-organization-of-polyribosomes-a-modern-approach
#15
REVIEW
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
https://www.readbyqxmd.com/read/29540525/a-microtubule-dynein-tethering-complex-regulates-the-axonemal-inner-dynein-f-i1
#16
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
https://www.readbyqxmd.com/read/29526767/advances-in-cryo-electron-tomography-for-biology-and-medicine
#17
REVIEW
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...
May 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
https://www.readbyqxmd.com/read/29519876/conserved-in-situ-arrangement-of-complex-i-and-iii-2-in-mitochondrial-respiratory-chain-supercomplexes-of-mammals-yeast-and-plants
#18
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 20, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29514928/the-i1-dynein-associated-tether-and-tether-head-complex-is-a-conserved-regulator-of-ciliary-motility
#19
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 cryoelectron tomography, we identified FAP44 and FAP43 (plus the algae-specific, FAP43-redundant FAP244) as T/TH components...
May 1, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29476122/author-correction-cryo-electron-tomography-reveals-that-dynactin-recruits-a-team-of-dyneins-for-processive-motility
#20
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.
April 2018: Nature Structural & Molecular Biology
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