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Cryo-EM

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https://www.readbyqxmd.com/read/29915388/cryo-em-structures-of-complex-i-from-mouse-heart-mitochondria-in-two-biochemically-defined-states
#1
Ahmed-Noor A Agip, James N Blaza, Hannah R Bridges, Carlo Viscomi, Shaun Rawson, Stephen P Muench, Judy Hirst
Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state...
June 18, 2018: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29915210/the-3-3-%C3%A3-structure-of-a-plant-geminivirus-using-cryo-em
#2
Emma L Hesketh, Keith Saunders, Chloe Fisher, Joran Potze, John Stanley, George P Lomonossoff, Neil A Ranson
Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of Ageratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves...
June 18, 2018: Nature Communications
https://www.readbyqxmd.com/read/29915197/conformational-switching-in-the-coiled-coil-domains-of-a-proteasomal-atpase-regulates-substrate-processing
#3
Aaron Snoberger, Evan J Brettrager, David M Smith
Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register...
June 18, 2018: Nature Communications
https://www.readbyqxmd.com/read/29915090/cryo-em-structure-of-human-mitochondrial-trifunctional-protein
#4
Kai Liang, Ningning Li, Xiao Wang, Jianye Dai, Pulan Liu, Chu Wang, Xiao-Wei Chen, Ning Gao, Junyu Xiao
The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane...
June 18, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29906447/helicase-dependent-rna-decay-illuminated-by-a-cryo-em-structure-of-a-human-nuclear-rna-exosome-mtr4-complex
#5
Eva-Maria Weick, M Rhyan Puno, Kurt Januszyk, John C Zinder, Michael A DiMattia, Christopher D Lima
The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM)...
June 14, 2018: Cell
https://www.readbyqxmd.com/read/29903714/new-insights-into-ribosome-structure-and-function
#6
Amy Jobe, Zheng Liu, Cristina Gutierrez-Vargas, Joachim Frank
In the past 4 years, because of the advent of new cameras, many ribosome structures have been solved by cryoelectron microscopy (cryo-EM) at high, often near-atomic resolution, bringing new mechanistic insights into the processes of translation initiation, peptide elongation, termination, and recycling. Thus, cryo-EM has joined X-ray crystallography as a powerful technique in structural studies of translation. The significance of this new development is that structures of ribosomes in complex with their functional binding partners can now be determined to high resolution in multiple states as they perform their work...
June 14, 2018: Cold Spring Harbor Perspectives in Biology
https://www.readbyqxmd.com/read/29899450/cryo-em-structure-of-human-rhodopsin-bound-to-an-inhibitory-g-protein
#7
Yanyong Kang, Oleg Kuybeda, Parker W de Waal, Somnath Mukherjee, Ned Van Eps, Przemyslaw Dutka, X Edward Zhou, Alberto Bartesaghi, Satchal Erramilli, Takefumi Morizumi, Xin Gu, Yanting Yin, Ping Liu, Yi Jiang, Xing Meng, Gongpu Zhao, Karsten Melcher, Oliver P Ernst, Anthony A Kossiakoff, Sriram Subramaniam, H Eric Xu
G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin...
June 13, 2018: Nature
https://www.readbyqxmd.com/read/29899147/conformational-control-and-dna-binding-mechanism-of-the-metazoan-origin-recognition-complex
#8
Franziska Bleichert, Alexander Leitner, Ruedi Aebersold, Michael R Botchan, James M Berger
In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2-7 (Mcm2-7) replicative helicase onto DNA to license origins. Drosophila ORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2-7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), we show that the autoinhibited state of Drosophila ORC is populated in solution, and that human ORC can also adopt this form...
June 13, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29887533/-cryo-electron-microscopy
#9
Atsuo Miyazawa
Cryo-electron microscopy (cryo-EM) includes three technical methods, (1) rapid freezing for vitreous ice-embedding, (2) observation of frozen hydrated specimens, and (3) image processing for three-dimensional structural analysis. The three-dimensional structural analysis can be performed in three different ways. Electron crystallography can decipher the structure of membrane proteins at the highest resolution (atomic level). Single particle analysis now allows the determiration of the structure of highly purified proteins and complexes (non-crystalline biomolecules) in solution at the near-atomic level...
June 2018: Brain and Nerve, Shinkei Kenkyū No Shinpo
https://www.readbyqxmd.com/read/29887376/structural-basis-for-transcript-elongation-control-by-nusg-family-universal-regulators
#10
Jin Young Kang, Rachel Anne Mooney, Yuri Nedialkov, Jason Saba, Tatiana V Mishanina, Irina Artsimovitch, Robert Landick, Seth A Darst
NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH...
May 30, 2018: Cell
https://www.readbyqxmd.com/read/29886737/molecular-mechanism-of-conductance-enhancement-in-narrow-cation-selective-membrane-channels
#11
Williams Ernesto Miranda, Van A Ngo, Ruiwu Wang, Lin Zhang, S R Wayne Chen, Sergei Yu Noskov
Membrane proteins known as Ryanodine Receptors (RyRs) display large-conductance of ~1 nS and nearly-ideal charge selectivity. Both properties are inversely correlated in other large-conductance but non-selective biological nanopores (i.e. alpha-hemolysin) used as industrial biosensors. Although recent Cryo-EM structures of RyR2 show similarities to K+ and Na+-selective channels, it remains unclear whether the similar ion conduction mechanisms occur in RyR2. Here, we combine micro-seconds of all-atom molecular dynamics (MD) simulations with mutagenesis and electrophysiology experiments to investigate large K+ conductance and charge (cation-vs-anion) selectivity in an open-state structure of RyR2...
June 10, 2018: Journal of Physical Chemistry Letters
https://www.readbyqxmd.com/read/29886014/structural-insights-into-the-role-of-diphthamide-on-elongation-factor-2-in-messenger-rna-reading-frame-maintenance
#12
Simone Pellegrino, Natalia Demeshkina, Eder Mancera-Martinez, Sergey Melnikov, Angelita Simonetti, Alexander Myasnikov, Marat Yusupov, Gulnara Yusupova, Yaser Hashem
One of the most critical steps of protein biosynthesis is the coupled movement of messenger RNA (mRNA), that encodes genetic information, with transfer RNAs (tRNAs) on the ribosome. In eukaryotes this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-EM structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights on the role of diphthamide in the mechanism of translation fidelity in eukaryotes...
June 7, 2018: Journal of Molecular Biology
https://www.readbyqxmd.com/read/29880918/cryo-em-in-drug-discovery-achievements-limitations-and-prospects
#13
REVIEW
Jean-Paul Renaud, Ashwin Chari, Claudio Ciferri, Wen-Ti Liu, Hervé-William Rémigy, Holger Stark, Christian Wiesmann
Cryo-electron microscopy (cryo-EM) of non-crystalline single particles is a biophysical technique that can be used to determine the structure of biological macromolecules and assemblies. Historically, its potential for application in drug discovery has been heavily limited by two issues: the minimum size of the structures it can be used to study and the resolution of the images. However, recent technological advances - including the development of direct electron detectors and more effective computational image analysis techniques - are revolutionizing the utility of cryo-EM, leading to a burst of high-resolution structures of large macromolecular assemblies...
June 8, 2018: Nature Reviews. Drug Discovery
https://www.readbyqxmd.com/read/29880725/structure-basis-for-rna-guided-dna-degradation-by-cascade-and-cas3
#14
Yibei Xiao, Min Luo, Adam E Dolan, Maofu Liao, Ailong Ke
Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA, by the RNA-guided Cascade complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here we present a 3.7 Å resolution cryo-EM structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the non-target strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA...
June 7, 2018: Science
https://www.readbyqxmd.com/read/29872539/cryo-em-structure-of-the-asic1a-mambalgin-1-complex-reveals-that-the-peptide-toxin-mambalgin-1-inhibits-acid-sensing-ion-channels-through-an-unusual-allosteric-effect
#15
Demeng Sun, You Yu, Xiaobin Xue, Man Pan, Ming Wen, Siyu Li, Qian Qu, Xiaorun Li, Longhua Zhang, Xueming Li, Lei Liu, Maojun Yang, Changlin Tian
Acid-sensing ion channels (ASICs) are neuronal voltage-independent Na+ channels that are activated by extracellular acidification. ASICs play essential roles in a wide range of physiological processes, including sodium homeostasis, synaptic plasticity, neurodegeneration, and sensory transduction. Mambalgins, a family of three-finger toxins isolated from black mamba venom, specifically inhibit ASICs to exert strong analgesic effects in vivo, thus are thought to have potential therapeutic values against pain...
2018: Cell Discovery
https://www.readbyqxmd.com/read/29872227/cryo-em-structure-of-the-human-neutral-amino-acid-transporter-asct2
#16
Alisa A Garaeva, Gert T Oostergetel, Cornelius Gati, Albert Guskov, Cristina Paulino, Dirk J Slotboom
Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses...
June 2018: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29872006/approaches-to-altering-particle-distributions-in-cryo-electron-microscopy-sample-preparation
#17
Ieva Drulyte, Rachel M Johnson, Emma L Hesketh, Daniel L Hurdiss, Charlotte A Scarff, Sebastian A Porav, Neil A Ranson, Stephen P Muench, Rebecca F Thompson
Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables...
June 1, 2018: Acta Crystallographica. Section D, Structural Biology
https://www.readbyqxmd.com/read/29872004/real-space-refinement-in-phenix-for-cryo-em-and-crystallography
#18
Pavel V Afonine, Billy K Poon, Randy J Read, Oleg V Sobolev, Thomas C Terwilliger, Alexandre Urzhumtsev, Paul D Adams
This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix...
June 1, 2018: Acta Crystallographica. Section D, Structural Biology
https://www.readbyqxmd.com/read/29872003/isolde-a-physically-realistic-environment-for-model-building-into-low-resolution-electron-density-maps
#19
Tristan Ian Croll
This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps. ISOLDE combines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints...
June 1, 2018: Acta Crystallographica. Section D, Structural Biology
https://www.readbyqxmd.com/read/29872001/current-approaches-for-the-fitting-and-refinement-of-atomic-models-into-cryo-em-maps-using-ccp-em
#20
Robert A Nicholls, Michal Tykac, Oleg Kovalevskiy, Garib N Murshudov
Recent advances in instrumentation and software have resulted in cryo-EM rapidly becoming the method of choice for structural biologists, especially for those studying the three-dimensional structures of very large macromolecular complexes. In this contribution, the tools available for macromolecular structure refinement into cryo-EM reconstructions that are available via CCP-EM are reviewed, specifically focusing on REFMAC5 and related tools. Whilst originally designed with a view to refinement against X-ray diffraction data, some of these tools have been able to be repurposed for cryo-EM owing to the same principles being applicable to refinement against cryo-EM maps...
June 1, 2018: Acta Crystallographica. Section D, Structural Biology
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