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cryo electron tomography

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https://www.readbyqxmd.com/read/28637765/electrostatic-interaction-between-polyglutamylated-tubulin-and-the-nexin-dynein-regulatory-complex-regulates-flagellar-motility
#1
Tomohiro Kubo, Toshiyuki Oda
Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme, and how tubulin polyglutamylation affects the flagellar motility. In this study, we identified the three-dimensional (3D) localization of the polyglutamylated tubulin in Chlamydomonas flagella using antibody labeling and cryo-electron tomography...
June 21, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28635493/the-structure-of-lamin-filaments-in-somatic-cells-as-revealed-by-cryo-electron-tomography
#2
Y Turgay, O Medalia
Metazoan nuclei are equipped with nuclear lamina - a thin layer of intermediate filaments (IFs) mostly built of nuclear lamins facing the inner nuclear membrane (INM). The nuclear lamina serves as an interaction hub for INM-proteins, soluble nuclear factors and DNA. It confers structural and mechanical stability to the nucleus, transduces mechanical forces and biochemical signals across the nuclear envelope (NE) and regulates the organization of chromatin. By utilizing cryo-electron tomography (cryo-ET), we recently provided an unprecedented view into the 3D organization of lamin filaments within the lamina meshwork in mammalian somatic cells...
June 21, 2017: Nucleus
https://www.readbyqxmd.com/read/28630150/cryo-electron-tomography-an-ideal-method-to-study-membrane-associated-proteins
#3
REVIEW
Michelle A Dunstone, Alex de Marco
Cryo-electron tomography (cryo-ET) is a three-dimensional imaging technique that makes it possible to analyse the structure of complex and dynamic biological assemblies in their native conditions. The latest technological and image processing developments demonstrate that it is possible to obtain structural information at nanometre resolution. The sample preparation required for the cryo-ET technique does not require the isolation of a protein and other macromolecular complexes from its native environment. Therefore, cryo-ET is emerging as an important tool to study the structure of membrane-associated proteins including pores...
August 5, 2017: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
https://www.readbyqxmd.com/read/28621666/9%C3%A3-structure-of-the-copi-coat-reveals-that-the-arf1-gtpase-occupies-two-contrasting-molecular-environments
#4
Svetlana O Dodonova, Patrick Aderhold, Juergen Kopp, Iva Ganeva, Simone Röhling, Wim J Hagen, Irmgard Sinning, Felix Wieland, John Ag Briggs
COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution...
June 16, 2017: ELife
https://www.readbyqxmd.com/read/28611252/the-primary-enveloped-virion-of-herpes-simplex-virus-1-its-role-in-nuclear-egress
#5
William W Newcomb, Juan Fontana, Dennis C Winkler, Naiqian Cheng, J Bernard Heymann, Alasdair C Steven
Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography...
June 13, 2017: MBio
https://www.readbyqxmd.com/read/28609782/influence-of-dna-sequence-on-the-structure-of-minicircles-under-torsional-stress
#6
Qian Wang, Rossitza N Irobalieva, Wah Chiu, Michael F Schmid, Jonathan M Fogg, Lynn Zechiedrich, B Montgomery Pettitt
The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence...
June 13, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28601688/structure-and-organization-of-paramyxovirus-particles
#7
REVIEW
Robert M Cox, Richard K Plemper
The paramyxovirus family comprises major human and animal pathogens such as measles virus (MeV), mumps virus (MuV), the parainfluenzaviruses, Newcastle disease virus (NDV), and the highly pathogenic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface. A number of crystal structures of different paramyxovirus proteins and protein fragments were solved, but the available information concerning overall virion organization remains limited...
June 8, 2017: Current Opinion in Virology
https://www.readbyqxmd.com/read/28580912/trends-in-the-electron-microscopy-data-bank-emdb
#8
Ardan Patwardhan
Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%...
June 1, 2017: Acta Crystallographica. Section D, Structural Biology
https://www.readbyqxmd.com/read/28575072/assembly-maturation-and-three-dimensional-helical-structure-of-the-teratogenic-rubella-virus
#9
Vidya Mangala Prasad, Thomas Klose, Michael G Rossmann
Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus...
June 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28559166/cryo-fib-specimen-preparation-for-use-in-a-cartridge-type-cryo-tem
#10
Jie He, Chyongere Hsieh, Yongping Wu, Thomas Schmelzer, Pan Wang, Ying Lin, Michael Marko, Haixin Sui
Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET...
May 27, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/28552576/tomominer-and-tomominercloud-a-software-platform-for-large-scale-subtomogram-structural-analysis
#11
Zachary Frazier, Min Xu, Frank Alber
Cryo-electron tomography (cryo-ET) captures the 3D electron density distribution of macromolecular complexes in close to native state. With the rapid advance of cryo-ET acquisition technologies, it is possible to generate large numbers (>100,000) of subtomograms, each containing a macromolecular complex. Often, these subtomograms represent a heterogeneous sample due to variations in the structure and composition of a complex in situ form or because particles are a mixture of different complexes. In this case subtomograms must be classified...
June 6, 2017: Structure
https://www.readbyqxmd.com/read/28551375/cytoskeletal-organization-in-microtentacles
#12
Alison N Killilea, Roseann Csencsits, Emily Le, Anand M Patel, Samuel J Kenny, Ke Xu, Kenneth H Downing
Microtentacles are thin, flexible cell protrusions that have recently been described and whose presence enhances efficient attachment of circulating cells. They are found on circulating tumor cells and can be induced on a wide range of breast cancer cell lines, where they are promoted by factors that either stabilize microtubules or destabilize the actin cytoskeleton. Evidence suggests that they are relevant to the metastatic spread of cancer, so understanding their structure and formation may lead to useful therapies...
May 24, 2017: Experimental Cell Research
https://www.readbyqxmd.com/read/28541401/three-dimensional-structural-labeling-microscopy-of-cilia-and-flagella
#13
Toshiyuki Oda
Locating a molecule within a cell using protein-tagging and immunofluorescence is a fundamental technique in cell biology, whereas in three-dimensional electron microscopy, locating a subunit within a macromolecular complex remains challenging. Recently, we developed a new structural labeling method for cryo-electron tomography by taking advantage of the biotin-streptavidin system, and have intensively used this method to locate a number of proteins and protein domains in cilia and flagella. In this review, we summarize our findings on the three-dimensional architecture of the axoneme, especially the importance of coiled-coil proteins...
May 25, 2017: Microscopy
https://www.readbyqxmd.com/read/28496159/a-comprehensive-guide-to-pilus-biogenesis-in-gram-negative-bacteria
#14
REVIEW
Manuela K Hospenthal, Tiago R D Costa, Gabriel Waksman
Pili are crucial virulence factors for many Gram-negative pathogens. These surface structures provide bacteria with a link to their external environments by enabling them to interact with, and attach to, host cells, other surfaces or each other, or by providing a conduit for secretion. Recent high-resolution structures of pilus filaments and the machineries that produce them, namely chaperone-usher pili, type IV pili, conjugative type IV secretion pili and type V pili, are beginning to explain some of the intriguing biological properties that pili exhibit, such as the ability of chaperone-usher pili and type IV pili to stretch in response to external forces...
May 12, 2017: Nature Reviews. Microbiology
https://www.readbyqxmd.com/read/28486768/the-trans-envelope-architecture-and-function-of-the-type-2-secretion-system-new-insights-raising-new-questions
#15
REVIEW
Jenny-Lee Thomassin, Javier Santos Moreno, Ingrid Guilvout, Guy Tran Van Nhieu, Olivera Francetic
Nanomachines belonging to the type IV filament (Tff) superfamily serve a variety of cellular functions in prokaryotes, including motility, adhesion, electrical conductance, competence and secretion. The type 2 secretion system (T2SS) Tff member assembles a short filament called pseudopilus that promotes the secretion of folded proteins from the periplasm across the outer membrane of Gram-negative bacteria. A combination of structural, biochemical, imaging, computational and in vivo approaches had led to a working model for the assembled nanomachine...
May 9, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28456019/dual-host-specificity-of-phage-sp6-is-facilitated-by-tailspike-rotation
#16
Jiagang Tu, Taehyun Park, Dustin R Morado, Kelly T Hughes, Ian J Molineux, Jun Liu
Bacteriophage SP6 exhibits dual-host adsorption specificity. The SP6 tailspikes are recognized as important in host range determination but the mechanisms underlying dual host specificity are unknown. Cryo-electron tomography and sub-tomogram classification were used to analyze the SP6 virion with a particular focus on the interaction of tailspikes with host membranes. The SP6 tail is surrounded by six V-shaped structures that interconnect in forming a hand-over-hand hexameric garland. Each V-shaped structure consists of two trimeric tailspike proteins: gp46 and gp47, connected through the adaptor protein gp37...
April 26, 2017: Virology
https://www.readbyqxmd.com/read/28412289/structural-studies-of-the-endogenous-spliceosome-the-supraspliceosome
#17
REVIEW
Joseph Sperling, Ruth Sperling
Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21MDa) and highly dynamic molecular machine - the supraspliceosome - that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA...
April 13, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28393831/determining-the-bacterial-cell-biology-of-planctomycetes
#18
Christian Boedeker, Margarete Schüler, Greta Reintjes, Olga Jeske, Muriel C F van Teeseling, Mareike Jogler, Patrick Rast, Daniela Borchert, Damien P Devos, Martin Kucklick, Miroslava Schaffer, Roberto Kolter, Laura van Niftrik, Susanne Engelmann, Rudolf Amann, Manfred Rohde, Harald Engelhardt, Christian Jogler
Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail...
April 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28389960/structure-of-the-sodium-driven-flagellar-motor-in-marine-vibrio
#19
Yasuhiro Onoue, Michio Homma
Most bacteria can swim by rotating the flagellum. The basal body of the flagellum is an essential part for this motor function. Recent comprehensive analysis of the flagellar basal body structures across bacteria by cryo-electron tomography has revealed that they all share core structures, the rod, and rings: the C ring, M ring, S ring, L ring, and P ring. Furthermore, it also has uncovered that in some bacteria, there are extra ring structures in the periplasmic space and outer-membrane. Here, we describe a protocol to isolate the basal body of the flagellar basal body from a marine bacterium, Vibrio alginolyticus, for structural analysis of additional ring structures, the T ring and H ring...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28389958/in-situ-structural-analysis-of-the-spirochetal-flagellar-motor-by-cryo-electron-tomography
#20
Shiwei Zhu, Zhuan Qin, Juyu Wang, Dustin R Morado, Jun Liu
The bacterial flagellar motor is a large multi-component molecular machine. Structural determination of such a large complex is often challenging and requires extensive structural analysis in situ. Cryo-electron tomography (cryo-ET) has emerged as a powerful technique that enables us to visualize intact flagellar motors in cells with unprecedented details. Here, we detail the procedure beginning with sample preparation, followed by data acquisition, tomographic reconstruction, sub-tomogram analysis, and ultimately visualization of the intact spirochetal flagellar motor in Borrelia burgdorferi...
2017: Methods in Molecular Biology
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