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cryo electron tomography

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https://www.readbyqxmd.com/read/27849173/imaging-the-motility-and-chemotaxis-machineries-in-helicobacter-pylori-by-cryo-electron-tomography
#1
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-ultra-oligotrophic-marine-bacterium-pelagibacter
#2
Xiaowei Zhao, Cindi Schwartz, Jason Pierson, Stephen J Giovannoni, J Richard McIntosh, Daniela Nicastro
: SAR11 bacteria are small, heterotrophic, marine α-proteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under these conditions are not well understood. To illuminate this issue we used cryo-electron tomography to study Pelagibacter, strain HTCC1062, a member of SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. The frozen-hydrated cells, which are preserved in a life-like state, have an average cell volume (enclosed by outer membrane) of 0...
November 11, 2016: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/27824088/trehalose-prevents-aggregation-of-exosomes-and-cryodamage
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
https://www.readbyqxmd.com/read/27688057/using-electron-microscopes-to-look-into-the-lung
#9
Matthias Ochs, Lars Knudsen, Jan Hegermann, Christoph Wrede, Roman Grothausmann, Christian Mühlfeld
In the nineteenth century, there was a dispute about the existence of a lung alveolar epithelium which remained unsolved until the invention of electron microscopy (EM) and its application to the lung. From the early 1960s, Ewald Weibel became the master of lung EM. He showed that the alveolar epithelium is covered with a lining layer containing surfactant. Weibel also explained the phenomenon of "non-nucleated plates" observed already in 1881 by Albert Kölliker. Weibel's most significant contribution was to the development of stereological methods...
September 29, 2016: Histochemistry and Cell Biology
https://www.readbyqxmd.com/read/27685097/resolving-macromolecular-structures-from-electron-cryo-tomography-data-using-subtomogram-averaging-in-relion
#10
Tanmay A M Bharat, Sjors H W Scheres
Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis...
November 2016: Nature Protocols
https://www.readbyqxmd.com/read/27678408/exploring-the-theoretical-basis-and-limitations-of-cryo-stem-tomography-for-thick-biological-specimens
#11
Peter Rez, Thomas Larsen, Michael Elbaum
Scanning transmission electron microscope (STEM) imaging has recently been applied to the cryo-tomography of thick biological specimens. As previously shown for plastic sections, STEM has a number of advantages for cryo-imaging compared to conventional wide-field TEM imaging. STEM is insensitive to phase coherence and is therefore suitable for much thicker specimens than TEM. Imaging in focus, with a long depth of field, also circumvents the complications of an oscillatory contrast transfer function and missing information at low spatial frequencies...
December 2016: Journal of Structural Biology
https://www.readbyqxmd.com/read/27671779/cryo-electron-tomography-can-it-reveal-the-molecular-sociology-of-cells-in-atomic-detail
#12
Martin Beck, Wolfgang Baumeister
Traditionally, macromolecular structure determination is performed ex situ, that is, with purified materials. But, there are strong incentives to develop approaches to study them in situ in their native functional context. In recent years, cryo-electron tomography (cryo-ET) has emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent work on several macromolecular assemblies, demonstrating the power of in situ studies...
September 23, 2016: Trends in Cell Biology
https://www.readbyqxmd.com/read/27663685/imaging-complement-by-phase-plate-cryo-electron-tomography-from-initiation-to-pore-formation
#13
Thomas H Sharp, Frank G A Faas, Abraham J Koster, Piet Gros
Phase plates in cryo-electron tomography (cryoET) improve contrast, increasing the ability to discern separate molecules and molecular complexes in dense biomolecular environments. Here, we applied this new technology to the activation of the human complement system. Binding of C1 to antigen-antibody complexes initiates a cascade of proteolytic events that deposits molecules onto adjacent surfaces and terminates with the formation of membrane-attack-complex (MAC) pores in the targeted membranes. We imaged steps in this process using a Volta phase plate mounted on a Titan Krios equipped with a Falcon-II direct electron detector...
September 20, 2016: Journal of Structural Biology
https://www.readbyqxmd.com/read/27650953/in-vitro-assembly-of-ebola-virus-nucleocapsid-like-complex-expressed-in-e-coli
#14
Ruchao Peng, Tengfei Zhu, Babayemi Olawale Oladejo, Abednego Moki Musyoki, Yingzi Cui, Yi Shi, Peiyi Wang, George Fu Gao
Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood...
September 20, 2016: Protein & Cell
https://www.readbyqxmd.com/read/27650306/near-atomic-three-dimensional-mapping-for-site-specific-chemistry-of-superbugs
#15
Vahid R Adineh, Ross K W Marceau, Tony Velkov, Jian Li, Jing Fu
Emergence of multidrug resistant Gram-negative bacteria has caused a global health crisis and last-line class of antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, the acquisition of a high-resolution chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale...
October 14, 2016: Nano Letters
https://www.readbyqxmd.com/read/27617931/eb1-interacts-with-outwardly-curved-and-straight-regions-of-the-microtubule-lattice
#16
Audrey Guesdon, Franck Bazile, Rubén M Buey, Renu Mohan, Solange Monier, Ruddi Rodríguez García, Morgane Angevin, Claire Heichette, Ralph Wieneke, Robert Tampé, Laurence Duchesne, Anna Akhmanova, Michel O Steinmetz, Denis Chrétien
EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice...
October 2016: Nature Cell Biology
https://www.readbyqxmd.com/read/27614295/next-generation-electron-microscopy-in-autophagy-research
#17
James H Hurley, Eva Nogales
Autophagy is the process whereby cytosol, organelles, and inclusions are taken up in a double-membrane vesicle known as the autophagosome, and transported to the lysosome for destruction and recycling. Electron microscopy (EM) led to the discovery of autophagy in the 1950s and has been a central part of its characterization ever since. New capabilities in single particle EM studies of the autophagy machinery are beginning to provide exciting insights into the mechanisms of autophagosome initiation, growth, and substrate targeting...
September 7, 2016: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/27572837/the-hemifusion-structure-induced-by-influenza-virus-haemagglutinin-is-determined-by-physical-properties-of-the-target-membranes
#18
Petr Chlanda, Elena Mekhedov, Hang Waters, Cindi L Schwartz, Elizabeth R Fischer, Rolf J Ryham, Fredric S Cohen, Paul S Blank, Joshua Zimmerberg
Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus...
2016: Nature Microbiology
https://www.readbyqxmd.com/read/27572733/cryo-electron-tomography-and-subtomogram-averaging
#19
W Wan, J A G Briggs
Cryo-electron tomography (cryo-ET) allows 3D volumes to be reconstructed from a set of 2D projection images of a tilted biological sample. It allows densities to be resolved in 3D that would otherwise overlap in 2D projection images. Cryo-ET can be applied to resolve structural features in complex native environments, such as within the cell. Analogous to single-particle reconstruction in cryo-electron microscopy, structures present in multiple copies within tomograms can be extracted, aligned, and averaged, thus increasing the signal-to-noise ratio and resolution...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27538822/polyhedral-3d-structure-of-human-plasma-very-low-density-lipoproteins-by-individual-particle-cryo-electron-tomography1
#20
Yadong Yu, Yu-Lin Kuang, Dongsheng Lei, Xiaobo Zhai, Meng Zhang, Ronald M Krauss, Gang Ren
Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures...
October 2016: Journal of Lipid Research
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