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cryo electron tomography

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https://www.readbyqxmd.com/read/28227855/simultaneous-reconstruction-and-segmentation-with-the-mumford-shah-functional-for-electron-tomography
#1
Li Shen, Eric Todd Quinto, Shiqiang Wang, Ming Jiang, Li Shen, Eric Todd Quinto, Shiqiang Wang, Ming Jiang, Ming Jiang, Eric Todd Quinto, Shiqiang Wang, Li Shen
Electron micrography (EM) is an important method for determining the three-dimensional (3D) structure of macromolecular complexes and biological specimens. But there are several limitations such as poor signal-to-noise, limitation on range of tilt angles and sub-region subject to electron exposure, unintentional movements of the specimen, with EM systems that make the reconstruction procedure a severely ill-posed problem. A different choice of reconstruction method may lead to different results and create different additional artifacts in reconstructed images...
August 2016: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/28218252/dissecting-the-molecular-organization-of-the-translocon-associated-protein-complex
#2
Stefan Pfeffer, Johanna Dudek, Miroslava Schaffer, Bobby G Ng, Sahradha Albert, Jürgen M Plitzko, Wolfgang Baumeister, Richard Zimmermann, Hudson H Freeze, Benjamin D Engel, Friedrich Förster
In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients...
February 20, 2017: Nature Communications
https://www.readbyqxmd.com/read/28216551/microscopic-characterization-of-the-brazilian-giant-samba-virus
#3
Jason R Schrad, Eric J Young, Jônatas S Abrahão, Juliana R Cortines, Kristin N Parent
Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts...
February 14, 2017: Viruses
https://www.readbyqxmd.com/read/28112764/cryo-electron-tomography-investigation-of-serum-albumin-camouflaged-tobacco-mosaic-virus-nanoparticles
#4
Neetu M Gulati, Andrzej S Pitek, Nicole F Steinmetz, Phoebe L Stewart
Nanoparticles offer great potential in drug delivery and imaging, but shielding strategies are necessary to increase circulation time and performance. Structure-function studies are required to define the design rules to achieve effective shielding. With several formulations reaching clinical testing and approval, the ability to assess and detail nanoparticle formulations at the single particle level is becoming increasingly important. To address this need, we use cryo-electron tomography (cryo-ET) to investigate stealth-coated nanoparticles...
January 23, 2017: Nanoscale
https://www.readbyqxmd.com/read/28100648/multi-scale-structural-analysis-of-plant-er-pm-contact-sites
#5
Heather E McFarlane, Eun Kyoung Lee, Laura S van Bezouwen, Bradford Ross, Abel Rosado, A Lacey Samuels
Membrane contact sites (MCS) are recognized across eukaryotic systems as important nanostructures. Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCS) are involved in excitation-contraction coupling, signalling, and plant responses to stress. In this report, we perform a multi-scale structural analysis of Arabidopsis EPCS that combine live cell imaging, quantitative transmission electron microscopy (TEM), and electron tomography over a developmental gradient. To place EPCS in the context of the entire cortical ER, we examined GFP-HDEL in living cells over a developmental gradient, then SYT1-GFP was used as a specific marker of EPCS...
January 18, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/28052246/synucleins-have-multiple-effects-on-presynaptic-architecture
#6
Karina J Vargas, Nikolas Schrod, Taylor Davis, Ruben Fernandez-Busnadiego, Yumiko V Taguchi, Ulrike Laugks, Vladan Lucic, Sreeganga S Chandra
Synucleins (α, β, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization...
January 3, 2017: Cell Reports
https://www.readbyqxmd.com/read/27993658/membrane-assisted-viral-dna-ejection
#7
Isaac Santos-Pérez, Hanna M Oksanen, Dennis H Bamford, Felix M Goñi, David Reguera, Nicola G A Abrescia
Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle...
March 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/27981875/three-dimensional-imaging-of-drosophila-motor-synapses-reveals-ultrastructural-organizational-patterns
#8
Hong Zhan, Joseph Bruckner, Ziheng Zhang, Kate O'Connor-Giles
We combined cryo-preservation of intact Drosophila larvae and electron tomography with comprehensive segmentation of key features to reconstruct the complete ultrastructure of a model glutamatergic synapse in a near-to-native state. Presynaptically, we detail a complex network of filaments that connects and organizes synaptic vesicles. We link the complexity of this synaptic vesicle network to proximity to the active zone cytomatrix, consistent with the model that these protein structures function together to regulate synaptic vesicle pools...
September 2016: Journal of Neurogenetics
https://www.readbyqxmd.com/read/27980210/the-structure-and-flexibility-of-conical-hiv-1-capsids-determined-within-intact-virions
#9
Simone Mattei, Bärbel Glass, Wim J H Hagen, Hans-Georg Kräusslich, John A G Briggs
HIV-1 contains a cone-shaped capsid encasing the viral genome. This capsid is thought to follow fullerene geometry-a curved hexameric lattice of the capsid protein, CA, closed by incorporating 12 CA pentamers. Current models for core structure are based on crystallography of hexameric and cross-linked pentameric CA, electron microscopy of tubular CA arrays, and simulations. Here, we report subnanometer-resolution cryo-electron tomography structures of hexameric and pentameric CA within intact HIV-1 particles...
December 16, 2016: Science
https://www.readbyqxmd.com/read/27977021/correlated-fluorescence-microscopy-and-cryo-electron-tomography-of-virus-infected-or-transfected-mammalian-cells
#10
Cheri M Hampton, Joshua D Strauss, Zunlong Ke, Rebecca S Dillard, Jason E Hammonds, Eric Alonas, Tanay M Desai, Mariana Marin, Rachel E Storms, Fredrick Leon, Gregory B Melikyan, Philip J Santangelo, Paul W Spearman, Elizabeth R Wright
Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27956360/defining-the-hierarchical-organisation-of-collagen-vi-microfibrils-at-nanometre-to-micrometre-length-scales
#11
Alan R F Godwin, Tobias Starborg, Michael J Sherratt, Alan M Roseman, Clair Baldock
: Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues...
December 10, 2016: Acta Biomaterialia
https://www.readbyqxmd.com/read/27943193/3d-analysis-of-synaptic-ultrastructure-in-organotypic-hippocampal-slice-culture-by-high-pressure-freezing-and-electron-tomography
#12
Cordelia Imig, Benjamin H Cooper
Transmission electron microscopy serves as a valuable tool for synaptic structure-function analyses aimed at identifying morphological features or modifications associated with specific developmental stages or dysfunctional synaptic states. By utilizing cryo-preparation techniques to minimize the introduction of structural artifacts during sample preparation, and electron tomography to reconstruct the 3D ultrastructural architecture of a synapse, the spatial organization and morphological properties of synaptic organelles and subcompartments can be quantified with unparalleled precision...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27849173/imaging-the-motility-and-chemotaxis-machineries-in-helicobacter-pylori-by-cryo-electron-tomography
#13
Zhuan Qin, Wei-Ting Lin, Shiwei Zhu, Aime T Franco, Jun Liu
: Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here we employ cryo-electron tomography to reveal detailed structures of the H. pylori cell envelope including the sheathed flagella and chemotaxis arrays. Notably, H...
November 14, 2016: Journal of Bacteriology
https://www.readbyqxmd.com/read/27836840/three-dimensional-structure-of-the-ultraoligotrophic-marine-bacterium-candidatus-pelagibacter-ubique
#14
Xiaowei Zhao, Cindi L Schwartz, Jason Pierson, Stephen J Giovannoni, J Richard McIntosh, Daniela Nicastro
: SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization...
February 1, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/27824088/trehalose-prevents-aggregation-of-exosomes-and-cryodamage
#15
Steffi Bosch, Laurence de Beaurepaire, Marie Allard, Mathilde Mosser, Claire Heichette, Denis Chrétien, Dominique Jegou, Jean-Marie Bach
Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry...
November 8, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27748356/nanoimaging-granule-dynamics-and-subcellular-structures-in-activated-mast-cells-using-soft-x-ray-tomography
#16
Huan-Yuan Chen, Dapi Meng-Lin Chiang, Zi-Jing Lin, Chia-Chun Hsieh, Gung-Chian Yin, I-Chun Weng, Peter Guttermann, Stephan Werner, Katja Henzler, Gerd Schneider, Lee-Jene Lai, Fu-Tong Liu
Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation...
October 17, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27742578/hierarchical-detection-and-analysis-of-macromolecular-complexes-in-cryo-electron-tomograms-using-pyto-software
#17
Vladan Lučić, Rubén Fernández-Busnadiego, Ulrike Laugks, Wolfgang Baumeister
Molecular complexes, arguably the basic units carrying cellular function, can be visualized directly in their native environment by cryo-electron tomography. Here we describe a procedure for the detection of small, pleomorphic membrane-bound molecular complexes in cryo-tomograms by a hierarchical connectivity segmentation. Validation on phantom and real data showed above 90% true positive rates. This segmentation procedure is implemented in the Pyto software package, together with methods for quantitative characterization and classification of complexes detected by our segmentation procedure and for statistical analysis between experimental conditions...
December 2016: Journal of Structural Biology
https://www.readbyqxmd.com/read/27731339/ultrastructure-of-compacted-dna-in-cyanobacteria-by-high-voltage-cryo-electron-tomography
#18
Kazuyoshi Murata, Sayuri Hagiwara, Yoshitaka Kimori, Yasuko Kaneko
Some cyanobacteria exhibit compaction of DNA in synchrony with their circadian rhythms accompanying cell division. Since the structure is transient, it has not yet been described in detail. Here, we successfully visualize the ultrastructure of compacted DNA in the cyanobacterium Synechococcus elongatus PCC 7942 under rigorous synchronized cultivation by means of high-voltage cryo-electron tomography. In 3D reconstructions of rapidly frozen cells, the compacted DNA appears as an undulating rod resembling a eukaryotic condensed chromosome...
October 12, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27726354/peptide-directed-assembly-of-single-helical-gold-nanoparticle-superstructures-exhibiting-intense-chiroptical-activity
#19
Andrea D Merg, Jennifer C Boatz, Abhishek Mandal, Gongpu Zhao, Soumitra Mokashi-Punekar, Chong Liu, Xianting Wang, Peijun Zhang, Patrick C A van der Wel, Nathaniel L Rosi
Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAu(M-ox))2 (PEPAu(M-ox) = AYSSGAPPM(ox)PPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles...
October 11, 2016: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/27716029/simulating-cryo-electron-tomograms-of-crowded-cell-cytoplasm-for-assessment-of-automated-particle-picking
#20
Long Pei, Min Xu, Zachary Frazier, Frank Alber
BACKGROUND: Cryo-electron tomography is an important tool to study structures of macromolecular complexes in close to native states. A whole cell cryo electron tomogram contains structural information of all its macromolecular complexes. However, extracting this information remains challenging, and relies on sophisticated image processing, in particular for template-free particle extraction, classification and averaging. To develop these methods it is crucial to realistically simulate tomograms of crowded cellular environments, which can then serve as ground truth models for assessing and optimizing methods for detection of complexes in cell tomograms...
October 5, 2016: BMC Bioinformatics
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