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Cryo-EM

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https://www.readbyqxmd.com/read/28102751/extracellular-vesicles-from-activated-platelets-a-semiquantitative-cryo-electron-microscopy-and-immuno-gold-labeling-study
#1
Alain R Brisson, Sisareuth Tan, Romain Linares, Céline Gounou, Nicolas Arraud
Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm...
January 19, 2017: Platelets
https://www.readbyqxmd.com/read/28096349/structure-of-eukaryotic-cmg-helicase-at-a-replication-fork-and-implications-to-replisome-architecture-and-origin-initiation
#2
Roxana Georgescu, Zuanning Yuan, Lin Bai, Ruda de Luna Almeida Santos, Jingchuan Sun, Dan Zhang, Olga Yurieva, Huilin Li, Michael E O'Donnell
The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork...
January 17, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28094514/structural-model-of-the-tubular-assembly-of-the-rous-sarcoma-virus-capsid-protein
#3
Jaekyun Jeon, Xin Qiao, Ivan Hung, Alok K Mitra, Ambroise Desfosses, Daniel Huang, Peter L Gor'kov, Rebecca C Craven, Richard L Kingston, Zhehong Gan, Fangqiang Zhu, Bo Chen
The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly-complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid...
January 17, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28092368/molecular-insights-into-lipid-assisted-ca-2-regulation-of-the-trp-channel-polycystin-2
#4
Martin Wilkes, M Gregor Madej, Lydia Kreuter, Daniel Rhinow, Veronika Heinz, Silvia De Sanctis, Sabine Ruppel, Rebecca M Richter, Friederike Joos, Marina Grieben, Ashley C W Pike, Juha T Huiskonen, Elisabeth P Carpenter, Werner Kühlbrandt, Ralph Witzgall, Christine Ziegler
Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca(2+) signaling pathways. Malfunctioning Ca(2+) regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2SI), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2MI)...
January 16, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28092267/cryo-em-structure-of-the-atp-sensitive-potassium-channel-illuminates-mechanisms-of-assembly-and-gating
#5
Gregory M Martin, Craig Yoshioka, Emily A Rex, Jonathan F Fay, Qing Xie, Matthew R Whorton, James Z Chen, Show-Ling Shyng
KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0)...
January 16, 2017: ELife
https://www.readbyqxmd.com/read/28088125/computational-methods-for-analyzing-conformational-variability-of-macromolecular-complexes-from-cryo-electron-microscopy-images
#6
REVIEW
Slavica Jonić
Thanks to latest technical advances in cryo-electron microscopy (cryo-EM), structures of macromolecular complexes (viruses, ribosomes, etc.) are now often obtained at near-atomic resolution. Also, studies of conformational changes of complexes, in connection with their function, are gaining ground. Conformational variability analysis is usually done by classifying images in a number of discrete classes supposedly representing all conformational states present in the specimen. However, discrete classes cannot be meaningfully defined when the conformational change is continuous (the specimen contains a continuum of states instead of a few discrete states)...
January 11, 2017: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/28086931/the-release-and-trans-synaptic-transmission-of-tau-via-exosomes
#7
Yipeng Wang, Varun Balaji, Senthilvelrajan Kaniyappan, Lars Krüger, Stephan Irsen, Katharina Tepper, RamReddy Chandupatla, Walter Maetzler, Anja Schneider, Eckhard Mandelkow, Eva-Maria Mandelkow
BACKGROUND: Tau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages ("Braak stages"). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood...
January 13, 2017: Molecular Neurodegeneration
https://www.readbyqxmd.com/read/28076346/cryo-em-structure-of-a-human-spliceosome-activated-for-step-2-of-splicing
#8
Karl Bertram, Dmitry E Agafonov, Wen-Ti Liu, Olexandr Dybkov, Cindy L Will, Klaus Hartmuth, Henning Urlaub, Berthold Kastner, Holger Stark, Reinhard Lu Hrmann
Spliceosome rearrangements facilitated by RNA helicase Prp16 before catalytic step 2 of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after Prp16 action (C*). The architecture of the catalytic U2-U6 RNP core of the human C* spliceosome is highly similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated (by ~20 Å) from the catalytic centre, and its position close to the U6 snRNA ACAGA box is stabilised by interactions with the Prp8 RNase H-like and Prp17 WD40 domains...
January 11, 2017: Nature
https://www.readbyqxmd.com/read/28074040/potent-neutralization-of-hepatitis-a-virus-reveals-a-receptor-mimic-mechanism-and-the-receptor-recognition-site
#9
Xiangxi Wang, Ling Zhu, Minghao Dang, Zhongyu Hu, Qiang Gao, Shuai Yuan, Yao Sun, Bo Zhang, Jingshan Ren, Abhay Kotecha, Thomas S Walter, Junzhi Wang, Elizabeth E Fry, David I Stuart, Zihe Rao
Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface...
January 10, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28067918/structural-insights-into-the-secretin-translocation-channel-in-the-type-ii-secretion-system
#10
Zhaofeng Yan, Meng Yin, Dandan Xu, Yongqun Zhu, Xueming Li
The secretin GspD of the type II secretion system (T2SS) forms a channel across the outer membrane in Gram-negative bacteria to transport substrates from the periplasm to the extracellular milieu. The lack of an atomic-resolution structure of the GspD channel hinders the investigation of substrate translocation mechanism of T2SS. Here we report cryo-EM structures of two GspD channels (∼1 MDa), from Escherichia coli K12 and Vibrio cholerae, at ∼3 Å resolution. The structures reveal a pentadecameric channel architecture, wherein three rings of GspD N domains form the periplasmic channel...
January 9, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28067916/self-correcting-mismatches-during-high-fidelity-dna-replication
#11
Rafael Fernandez-Leiro, Julian Conrad, Ji-Chun Yang, Stefan M V Freund, Sjors H W Scheres, Meindert H Lamers
Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands...
January 9, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28067914/structure-of-the-immature-zika-virus-at-9-%C3%A3-resolution
#12
Vidya Mangala Prasad, Andrew S Miller, Thomas Klose, Devika Sirohi, Geeta Buda, Wen Jiang, Richard J Kuhn, Michael G Rossmann
The current Zika virus (ZIKV) epidemic is characterized by severe pathogenicity in both children and adults. Sequence changes in ZIKV since its first isolation are apparent when pre-epidemic strains are compared with those causing the current epidemic. However, the residues that are responsible for ZIKV pathogenicity are largely unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of the immature ZIKV at 9-Å resolution. The cryo-EM map was fitted with the crystal structures of the precursor membrane and envelope glycoproteins and was shown to be similar to the structures of other known immature flaviviruses...
January 9, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28065261/expression-purification-and-enzymatic-characterization-of-intramembrane-proteases
#13
R Zhou, Y Shi, G Yang
Intramembrane proteases catalyze peptide bond hydrolysis in the lipid bilayer and play a key role in numerous cellular processes. These integral membrane enzymes consist of four classes: site-2 protease (S2P), rhomboid serine protease, Rce1-type glutamyl protease, and aspartyl protease exemplified by presenilin and signal peptide peptidase (SPP). Structural elucidation of these enzymes is important for mechanistic understanding of their functions, particularly their roles in cell signaling and debilitating diseases such as Parkinson's disease and Alzheimer's disease...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28059769/cryo-em-structures-and-atomic-model-of-the-hiv-1-strand-transfer-complex-intasome
#14
Dario Oliveira Passos, Min Li, Renbin Yang, Stephanie V Rebensburg, Rodolfo Ghirlando, Youngmin Jeon, Nikoloz Shkriabai, Mamuka Kvaratskhelia, Robert Craigie, Dmitry Lyumkis
Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements...
January 6, 2017: Science
https://www.readbyqxmd.com/read/28055188/assembling-enzymatic-cascade-pathways-inside-virus-based-nanocages-using-dual-tasking-nucleic-acid-tags
#15
Melanie Brasch, Rindia M Putri, Mark Vincent de Ruiter, Daniel Luque, Melissa S T Koay, Jose R Castón, Jeroen J L M Cornelissen
The packaging of proteins into discrete compartments is an essential feature for cellular efficiency. Inspired by Nature, we harness virus-like assemblies as artificial nanocompartments for enzyme-catalyzed cascade reactions. Using the negative charges of nucleic acid tags, we develop a versatile strategy to promote an efficient non-covalent co-encapsulation of enzymes within a single protein cage of Cowpea Chlorotic Mottle Virus (CCMV) at neutral pH. The encapsulation results in stable 21-22 nm-sized CCMV-like particles, which is characteristic of an icosahedral T=1 symmetry...
January 5, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28055037/advances-in-the-field-of-single-particle-cryo-electron-microscopy-over-the-last-decade
#16
Joachim Frank
In single-particle cryo-electron microscopy (cryo-EM), molecules suspended in a thin aqueous layer are rapidly frozen and imaged at cryogenic temperature in the transmission electron microscope. From the random projection views, a three-dimensional image is reconstructed, enabling the structure of the molecule to be obtained. In this article I discuss technological progress over the past decade, which has, in my own field of study, culminated in the determination of ribosome structure at 2.5-Å resolution. I also discuss likely future improvements in methodology...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28053231/cryo-em-study-of-slow-bee-paralysis-virus-at-low-ph-reveals-iflavirus-genome-release-mechanism
#17
Sergei Kalynych, Tibor Füzik, Antonín Přidal, Joachim de Miranda, Pavel Plevka
Viruses from the family Iflaviridae are insect pathogens. Many of them, including slow bee paralysis virus (SBPV), cause lethal diseases in honeybees and bumblebees, resulting in agricultural losses. Iflaviruses have nonenveloped icosahedral virions containing single-stranded RNA genomes. However, their genome release mechanism is unknown. Here, we show that low pH promotes SBPV genome release, indicating that the virus may use endosomes to enter host cells. We used cryo-EM to study a heterogeneous population of SBPV virions at pH 5...
January 17, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28049779/cryoelectron-microscopy-of-fission-yeast
#18
Mary K Morphew, Thomas H Giddings, J Richard McIntosh
Fission yeast cells can be prepared for electron microscopy (EM) in the frozen-hydrated state. This eliminates the requirement for dehydration and heavy metal staining when preparing samples for EM. As with room temperature imaging, however, the yeast must be sectioned to make them thin enough for transmission of the electron beam. Cutting sections of vitreous ice with a microtome is challenging. An alternative method that uses a focused ion beam to make a thin sample by milling away much of the sample at liquid nitrogen temperatures is under development but is not yet available for routine use...
January 3, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28042333/nanosized-ucmsc-derived-extracellular-vesicles-but-not-conditioned-medium-exclusively-inhibit-the-inflammatory-response-of-stimulated-t-cells-implications-for-nanomedicine
#19
Marta Monguió-Tortajada, Santiago Roura, Carolina Gálvez-Montón, Josep Maria Pujal, Gemma Aran, Lucía Sanjurjo, Marcel la Franquesa, Maria-Rosa Sarrias, Antoni Bayes-Genis, Francesc E Borràs
Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data...
2017: Theranostics
https://www.readbyqxmd.com/read/28024108/a-snapshot-of-cryo-em
#20
EDITORIAL
Georgios Skiniotis
No abstract text is available yet for this article.
January 2017: Protein Science: a Publication of the Protein Society
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