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cryo EM

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https://www.readbyqxmd.com/read/29220409/acquisition-of-functions-on-the-outer-capsid-surface-during-evolution-of-double-stranded-rna-fungal-viruses
#1
Carlos P Mata, Daniel Luque, Josué Gómez-Blanco, Javier M Rodríguez, José M González, Nobuhiro Suzuki, Said A Ghabrial, José L Carrascosa, Benes L Trus, José R Castón
Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the ~450-Å-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues...
December 8, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/29218561/applications-of-sans-to-study-membrane-protein-systems
#2
Frank Gabel
Small angle neutron scattering (SANS) is a powerful tool to obtain structural information on solubilized membrane proteins on the nanometer length-scale in complement to other structural biology techniques such as cryo-EM, NMR and SAXS. In combination with deuteration of components and/or contrast variation (H2O:D2O exchange in the buffer) SANS allows to separate structural information from the protein and the detergent/lipid parts in solution. After a short historical overview on results obtained by SANS on membrane protein systems, this book chapter introduces the basic theoretical principles of the technique as well as requirements on samples...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/29215632/assembly-of-phospholipid-nanodiscs-of-controlled-size-for-structural-studies-of-membrane-proteins-by-nmr
#3
Franz Hagn, Mahmoud L Nasr, Gerhard Wagner
Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29214692/cryo-em-revolutionizes-the-structure-determination-of-biomolecules
#4
Stefan Raunser
Freeze frame: The Nobel Prize in Chemistry 2017 was jointly awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution". This Highlight describes how the field of cryo-EM developed from the first transmission electron microscope in 1931 to the sophisticated technique of today.
December 7, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/29212814/structural-basis-for-assembly-of-the-cbf3-kinetochore-complex
#5
Vera Leber, Andrea Nans, Martin R Singleton
Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP-A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP-ACse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single-particle cryo-EM reconstruction of the core CBF3 complex, incorporating the sequence-specific DNA-binding protein Cep3 together with regulatory subunits Ctf13 and Skp1...
December 6, 2017: EMBO Journal
https://www.readbyqxmd.com/read/29211983/how-and-why-chaperone-usher-pilus-rods-stretch
#6
Hye-Jeong Yeo
Type 1 and P pili are important virulence factors of uropathogenic Escherichia coli, the leading cause of urinary tract infections. In this issue of Structure, Hospenthal et al. (2017) describe a near-atomic resolution cryo-EM structure of the type 1 pilus rod, providing molecular insights into rod uncoiling in two pili.
December 5, 2017: Structure
https://www.readbyqxmd.com/read/29203665/structural-studies-of-chikungunya-virus-maturation
#7
Moh Lan Yap, Thomas Klose, Akane Urakami, S Saif Hasan, Wataru Akahata, Michael G Rossmann
Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an "immature" Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B...
December 4, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29202442/molecular-simulations-of-the-ribosome-and-associated-translation-factors
#8
REVIEW
Lars V Bock, Michal H Kolář, Helmut Grubmüller
The ribosome is a macromolecular complex which is responsible for protein synthesis in all living cells according to their transcribed genetic information. Using X-ray crystallography and, more recently, cryo-electron microscopy (cryo-EM), the structure of the ribosome was resolved at atomic resolution in many functional and conformational states. Molecular dynamics simulations have added information on dynamics and energetics to the available structural information, thereby have bridged the gap to the kinetics obtained from single-molecule and bulk experiments...
December 1, 2017: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/29199952/cryo-em-structures-reveal-specialization-at-the-myosin-vi-actin-interface-and-a-mechanism-of-force-sensitivity
#9
Pinar S Gurel, Laura Y Kim, Paul V Ruijgrok, Tosan Omabegho, Zev Bryant, Gregory M Alushin
Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Å) and Mg-ADP (5.5 Å) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization...
December 4, 2017: ELife
https://www.readbyqxmd.com/read/29196491/diversity-of-nicotinic-acetylcholine-receptor-positive-allosteric-modulators-revealed-by-mutagenesis-and-a-revised-structural-model
#10
Joseph Newcombe, Anna Chatzidaki, Tom D Sheppard, Maya Topf, Neil S Millar
By combining electrophysiological and computational approaches we have examined a series of positive allosteric modulators (PAMs) acting on the human α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological studies have focussed on three α7-selective PAMs (A-867744, TBS-516 and TQS) that display similar effects on wild-type α7 nAChRs. In addition to potentiating agonist-evoked responses, all three compounds reduce receptor desensitisation and, consequently, are classed as type II PAMs. Despite having similar effects on wild-type receptors, A-867744 was found to have profoundly differing effects to TBS-516 and TQS on mutated receptors, a finding that is consistent with previous studies indicating that A-867744 may have a different mechanism of action to other α7-selective type II PAMs...
December 1, 2017: Molecular Pharmacology
https://www.readbyqxmd.com/read/29195065/how-cryo-em-became-so-hot
#11
Yifan Cheng, Robert M Glaeser, Eva Nogales
The Royal Swedish Academy of Sciences awarded the 2017 Nobel Prize for Chemistry to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryoelectron microscopy for the high-resolution structure determination of biomolecules in solution." Achieving this goal, which required innovation, persistence, and uncommon physical insight, has broadened horizons for structural studies in molecular and cell biology.
November 30, 2017: Cell
https://www.readbyqxmd.com/read/29191673/automated-tracing-of-helical-assemblies-from-electron-cryo-micrographs
#12
Stefan Huber, Tanja Kuhm, Carsten Sachse
Structure determination of helical specimens commonly requires datasets from thousands of micrographs often obtained by automated cryo-EM data acquisition. Interactive tracing of helical assemblies from such a number of micrographs is labor-intense and time-consuming. Here, we introduce an automated tracing tool MicHelixTrace that precisely locates helix traces from micrographs of rigid as well as very flexible helical assemblies with small numbers of false positives. The computer program is fast and has low computational requirements...
November 27, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/29187545/novel-insect-specific-eilat-virus-based-chimeric-vaccine-candidates-provide-durable-mono-and-multi-valent-single-dose-protection-against-lethal-alphavirus-challenge
#13
Jesse H Erasmus, Robert L Seymour, Jason T Kaelber, Dal Y Kim, Grace Leal, Michael B Sherman, Ilya Frolov, Wah Chiu, Scott C Weaver, Farooq Nasar
Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals...
November 29, 2017: Journal of Virology
https://www.readbyqxmd.com/read/29177826/purification-of-acrab-tolc-multidrug-efflux-pump-for-cryo-em-analysis
#14
Dijun Du, Zhao Wang, Wah Chiu, Ben F Luisi
The cell envelope of Gram-negative bacteria comprises an outer membrane, a cytoplasmic inner membrane, and an interstitial space. The tripartite multidrug transporter AcrAB-TolC, which uses proton electrochemical gradients to vectorially drive the efflux of drugs from the cell, spans this envelope. We describe here details of the methods used to prepare the recombinant tripartite assembly for high-resolution structure determination by cryo-EM.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29176698/the-development-of-cryo-em-and-how-it-has-advanced-microbiology
#15
Catherine M Oikonomou, Grant J Jensen
No abstract text is available yet for this article.
December 2017: Nature Microbiology
https://www.readbyqxmd.com/read/29176596/quenching-protein-dynamics-interferes-with-hiv-capsid-maturation
#16
Mingzhang Wang, Caitlin M Quinn, Juan R Perilla, Huilan Zhang, Randall Shirra, Guangjin Hou, In-Ja Byeon, Christopher L Suiter, Sherimay Ablan, Emiko Urano, Theodore J Nitz, Christopher Aiken, Eric O Freed, Peijun Zhang, Klaus Schulten, Angela M Gronenborn, Tatyana Polenova
Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA-SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA-SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix-coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1...
November 24, 2017: Nature Communications
https://www.readbyqxmd.com/read/29175288/mrcz-a-file-format-for-cryo-tem-data-with-fast-compression
#17
Robert A McLeod, Ricardo Diogo Righetto, Andy Stewart, Henning Stahlberg
The introduction of fast CMOS detectors is moving the field of transmission electron microscopy into the computer science field of big data. Automated data pipelines control the instrument and initial processing steps which imposes more onerous data transfer and archiving requirements. Here we conduct a technical demonstration whereby storage and read/write times are improved 10x at a dose rate of 1 e-/pix/frame for data from a Gatan K2 direct-detection device by combination of integer decimation and lossless compression...
November 23, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/29171028/the-mechanosensitive-piezo1-channel-structural-features-and-molecular-bases-underlying-its-ion-permeation-and-mechanotransduction
#18
Yubo Wang, Bailong Xiao
The evolutionarily conserved Piezo family of proteins, including Piezo1 and Piezo2, encodes the long-sought-after mammalian mechanosensitive cation channels that play critical roles in various mechanotransduction processes such as touch, pain, proprioception, vascular development and blood pressure regulation. Mammalian Piezo proteins contain over 2500 amino acids with numerous predicted transmembrane segments, and do not bear sequence homology with any known class of ion channels. Thus, it is imperative, but challenging, to understand how they serve as effective mechanotransducers for converting mechanical force into electrochemical signals...
November 24, 2017: Journal of Physiology
https://www.readbyqxmd.com/read/29170376/cryo-em-structure-of-saccharomyces-cerevisiae-target-of-rapamycin-complex-2
#19
Manikandan Karuppasamy, Beata Kusmider, Taiana M Oliveira, Christl Gaubitz, Manoel Prouteau, Robbie Loewith, Christiane Schaffitzel
The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins...
November 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/29167449/structure-of-human-immunoproteasome-with-a-reversible-and-noncompetitive-inhibitor-that-selectively-inhibits-activated-lymphocytes
#20
Ruda de Luna Almeida Santos, Lin Bai, Pradeep K Singh, Naoka Murakami, Hao Fan, Wenhu Zhan, Yingrong Zhu, Xiuju Jiang, Kaiming Zhang, Jean Pierre Assker, Carl F Nathan, Huilin Li, Jamil Azzi, Gang Lin
Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively, an AsnEDA inhibitor binds the active site...
November 22, 2017: Nature Communications
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