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cryo electron tomography

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https://www.readbyqxmd.com/read/28332496/cell-free-reconstitution-reveals-centriole-cartwheel-assembly-mechanisms
#1
P Guichard, V Hamel, M Le Guennec, N Banterle, I Iacovache, V Nemčíková, I Flückiger, K N Goldie, H Stahlberg, D Lévy, B Zuber, P Gönczy
How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly...
March 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28330771/deciphering-the-molecular-architecture-of-membrane-contact-sites-by-cryo-electron-tomography
#2
REVIEW
Javier Collado, Rubén Fernández-Busnadiego
At membrane contact sites (MCS) two cellular membranes form tight appositions that play critical roles in fundamental phenomena such as lipid metabolism or Ca(2+) homeostasis. The interest for these structures has surged in recent years, bringing about the characterization of a plethora of MCS-resident molecules. How those molecules are structurally organized at MCS remains enigmatic, limiting our understanding of MCS function. Whereas such molecular detail is obscured by conventional electron microscopy sample preparation, cryo-electron tomography (cryo-ET) is uniquely positioned to address this challenge by allowing high resolution imaging of cellular landscapes in close-to-native conditions...
March 19, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28323617/cryoem-structures-of-membrane-pore-and-prepore-complex-reveal-cytolytic-mechanism-of-pneumolysin
#3
Katharina van Pee, Alexander Neuhaus, Edoardo D'Imprima, Deryck J Mills, Werner Kühlbrandt, Özkan Yildiz
Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain D3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel...
March 21, 2017: ELife
https://www.readbyqxmd.com/read/28291359/challenges-of-integrating-stochastic-dynamics-and-cryo-electron-tomograms-in-whole-cell-simulations
#4
Tyler M Earnest, Reika Watanabe, John E Stone, Julia Mahamid, Wolfgang Baumeister, Elizabeth Villa, Zaida Luthey-Schulten
Cryo-electron tomography (cryo-ET) has rapidly emerged as a powerful tool to investigate the internal, three-dimensional spatial organization of the cell. In parallel, the GPU-based technology to perform spatially resolved stochastic simulations of whole cells has arisen, allowing the simulation of complex biochemical networks over cell cycle timescales using data taken from -omics, single molecule experiments, and in vitro kinetics. By using real cell geometry derived from cryo-ET data, we have the opportunity to imbue these highly detailed structural data-frozen in time-with realistic biochemical dynamics and investigate how cell structure affects the behavior of the embedded chemical reaction network...
March 14, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28283066/inside-the-chamber-of-secrets-of-the-type-iii-secretion-system
#5
Eric Cascales
The bacterial type III secretion system is a specialized machine that injects effectors into eukaryotic cells to manipulate the host cell physiology. In this issue of Cell, Hu et al. use cryo-electron tomography to reveal an unprecedented level of details regarding the architecture of this machine and the conformational changes that occur during its assembly.
March 9, 2017: Cell
https://www.readbyqxmd.com/read/28283062/in-situ-molecular-architecture-of-the-salmonella-type-iii-secretion-machine
#6
Bo Hu, Maria Lara-Tejero, Qingke Kong, Jorge E Galán, Jun Liu
Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components...
March 9, 2017: Cell
https://www.readbyqxmd.com/read/28281671/visualizing-adsorption-of-cyanophage-p-ssp7-onto-marine-prochlorococcus
#7
Kazuyoshi Murata, Qinfen Zhang, Jesús Gerardo Galaz-Montoya, Caroline Fu, Maureen L Coleman, Marcia S Osburne, Michael F Schmid, Matthew B Sullivan, Sallie W Chisholm, Wah Chiu
Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events...
March 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28276027/analysis-of-mitochondrial-membrane-protein-complexes-by-electron-cryo-tomography
#8
Vicki A M Gold, Tobias Brandt, Laetitia Cavellini, Mickael M Cohen, Raffaele Ieva, Martin van der Laan
The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28269599/simultaneous-reconstruction-and-segmentation-with-the-mumford-shah-functional-for-electron-tomography
#9
Li Shen, Eric Todd Quinto, Shiqiang Wang, Ming Jiang
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/28241138/the-molecular-architecture-of-lamins-in-somatic-cells
#10
Yagmur Turgay, Matthias Eibauer, Anne E Goldman, Takeshi Shimi, Maayan Khayat, Kfir Ben-Harush, Anna Dubrovsky-Gaupp, K Tanuj Sapra, Robert D Goldman, Ohad Medalia
The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina...
March 1, 2017: Nature
https://www.readbyqxmd.com/read/28227855/simultaneous-reconstruction-and-segmentation-with-the-mumford-shah-functional-for-electron-tomography
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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