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Single particle electron microscopy

Christian Beren, Lisa L Dreesens, Katherine N Liu, Charles M Knobler, William M Gelbart
Previous work has shown that purified capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) is capable of packaging both purified single-stranded RNA molecules of normal composition (comparable numbers of A, U, G, and C nucleobases) and of varying length and sequence, and anionic synthetic polymers such as polystyrene sulfonate. We find that CCMV CP is also capable of packaging polyU RNAs, which-unlike normal-composition RNAs-do not form secondary structures and which act as essentially structureless linear polymers...
July 12, 2017: Biophysical Journal
Jamie I Vandenberg, Eduardo Perozo, Toby W Allen
The human ether-a-go-go-related gene (hERG) K(+) channel is of great medical and pharmaceutical relevance. Inherited mutations in hERG result in congenital long-QT syndrome which is associated with a markedly increased risk of cardiac arrhythmia and sudden death. hERG K(+) channels are also remarkably susceptible to block by a wide range of drugs, which in turn can cause drug-induced long-QT syndrome and an increased risk of sudden death. The recent determination of the near-atomic resolution structure of the hERG K(+) channel, using single-particle cryo-electron microscopy (cryo-EM), provides tremendous insights into how these channels work...
July 12, 2017: Trends in Pharmacological Sciences
Soung-Hun Roh, Corey F Hryc, Hyun-Hwan Jeong, Xue Fei, Joanita Jakana, George H Lorimer, Wah Chiu
Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations...
July 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
Takashi Hirama, Raibatak Das, Yanbo Yang, Charles Ferguson, Amy Won, Christopher M Yip, Jason G Kay, Sergio Grinstein, Robert G Parton, Gregory D Fairn
Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (CAVs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) on caveolar formation and dynamics...
July 11, 2017: Journal of Biological Chemistry
Priyanka D Abeyrathne, Nikolaus Grigorieff
Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis...
2017: PloS One
Jie Zhang, Bartosz A Grzybowski, Steve Granick
Janus particles are colloidal particles with more than a single type of surface chemistry or composition, ranging in size from hundreds of nanometers to a few micrometers. Like traditional colloids, they are large enough to be observed under optical microscopy in real time and small enough to diffuse by Brownian motion, but their interesting and useful new properties of directional interaction bring new research opportunities to the fields of soft matter and fundamental materials research as well as to applications in other disciplines and in technologies such as electronic paper and other multiphase engineering...
July 5, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Radostin Danev, Wolfgang Baumeister
Phase plates have long been considered as a means for improving the performance of cryo-electron microscopy (cryo-EM). But practical limitations, such as a short lifespan or cumbersome usage have prevented their widespread adoption. The recently developed Volta phase plate overcomes most of the practical issues and it is now commercially available. Results from both, electron cryo-tomography (cryo-ET) and single particle analysis (SPA), have demonstrated the benefits of using a phase plate. In CET phase plates have helped to visualize cellular ultrastructure in unprecedented detail...
July 1, 2017: Current Opinion in Structural Biology
Yong Zi Tan, Philip R Baldwin, Joseph H Davis, James R Williamson, Clinton S Potter, Bridget Carragher, Dmitry Lyumkis
We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations...
July 3, 2017: Nature Methods
Ramaswamy Shanmugam, Dwarampudi L Priyanka, Kadiyala Madhuri, Kuppuswamy Gowthamarajan, Veera Venkata Satyanarayana Reddy Karri, Chivarama K Ashok Kumar, Paranjothy Murali
Curcumin and Rutin are natural polyphenolic molecules exhibits several pharmacological actives like antibacterial, anticancer, antioxidant, chemo-preventive and anti-inflammatory properties. However till date, no studies have been reported on their combination efficacy, especially in treating multi-drug resistance of cancers because of their poor solubility and bioavailability. Hence in the present study, an attempt has been made to load both these drugs into a single nanoparticlulate system to enhance their bioavailability and efficacy...
June 28, 2017: International Journal of Biological Macromolecules
Ralf Ficner, Achim Dickmanns, Piotr Neumann
The splicing of eukaryotic precursor mRNAs requires the activity of at least three DEAD-box helicases, one Ski2-like helicase and four DEAH-box helicases. High resolution structures for five of these spliceosomal helicases were obtained by means of X-ray crystallography. Additional low resolution structural information could be derived from single particle cryo electron microscopy and small angle X-ray scattering. The functional characterization includes biochemical methods to measure the ATPase and helicase activities...
June 28, 2017: Methods: a Companion to Methods in Enzymology
Marta Sawicka, Ricardo Aramayo, Rafael Ayala, Robert Glyde, Xiaodong Zhang
DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein production yield, a conformational flexibility, and a relatively high molecular mass. Single-particle electron microscopy (EM) has been successfully applied to study some of these complexes as it requires low amount of samples, is not limited by the high molecular mass of a protein or a complex, and can separate heterogeneous assemblies...
2017: Methods in Enzymology
Tiago R D Costa, Athanasios Ignatiou, Elena V Orlova
Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011)...
2017: Methods in Molecular Biology
Miki Nakano, Osamu Miyashita, Slavica Jonic, Changyong Song, Daewoong Nam, Yasumasa Joti, Florence Tama
The three-dimensional (3D) structural analysis of single particles using an X-ray free-electron laser (XFEL) is a new structural biology technique that enables observations of molecules that are difficult to crystallize, such as flexible biomolecular complexes and living tissue in the state close to physiological conditions. In order to restore the 3D structure from the diffraction patterns obtained by the XFEL, computational algorithms are necessary as the orientation of the incident beam with respect to the sample needs to be estimated...
July 1, 2017: Journal of Synchrotron Radiation
Kaiser Leung, Elijah G Schnitzler, Ramin Dastanpour, Steven Rogak, Wolfgang Jaeger, Jason S Olfert
The restructuring of mono-disperse soot aggregates due to coatings of secondary organic aerosol (SOA) was investigated in a series of photo-oxidation chamber experiments. Soot aggregates were generated by one of three sources (an ethylene premixed burner, a methane inverted diffusion burner, or a diesel generator), treated by denuding, size-selected by a differential mobility analyzer, and injected into a smog chamber, where they were exposed to the photo-oxidation products of p-xylene, which partitioned to form SOA coatings...
June 29, 2017: Environmental Science & Technology
Peng Jin, David Bulkley, Yanmeng Guo, Wei Zhang, Zhenhao Guo, Walter Huynh, Shenping Wu, Shan Meltzer, Tong Cheng, Lily Yeh Jan, Yuh-Nung Jan, Yifan Cheng
Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels...
June 26, 2017: Nature
Max J Schnepf, Martin Mayer, Christian Kuttner, Moritz Tebbe, Daniel Wolf, Martin Dulle, Thomas Altantzis, Petr Formanek, Stephan Förster, Sara Bals, Tobias A F König, Andreas Fery
Nanorattles are metallic core-shell particles with core and shell separated by a dielectric spacer. These nanorattles have been identified as a promising class of nanoparticles, due to their extraordinary high electric-field enhancement inside the cavity. Limiting factors are reproducibility and loss of axial symmetry owing to the movable metal core; movement of the core results in fluctuation of the nanocavity dimensions and commensurate variations in enhancement factor. We present a novel synthetic approach for the robust fixation of the central gold rod within a well-defined box, which results in an axisymmetric nanorattle...
July 13, 2017: Nanoscale
Sang Tae Park, Renske M van der Veen
In this article, we present a continuum mechanics based approach for modeling thermally induced single-nanoparticle phase transitions studied in ultrafast electron microscopy. By using coupled differential equations describing heat transfer and the kinetics of the phase transition, we determine the major factors governing the time scales and efficiencies of thermal switching in individual spin-crossover nanoparticles, such as the thermal properties of the (graphite) substrate, the particle thickness, and the interfacial thermal contact conductance between the substrate and the nanoparticle...
July 2017: Structural Dynamics (Melville, N.Y.)
Dheva T Setiaputra, Calvin K Yip
Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This organization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated factors...
June 23, 2017: Biochimica et Biophysica Acta
George G Daaboul, David S Freedman, Steven M Scherr, Erik Carter, Alexandru Rosca, David Bernstein, Chad E Mire, Krystle N Agans, Thomas Hoenen, Thomas W Geisbert, M Selim Ünlü, John H Connor
Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view...
2017: PloS One
Geoffrey Masuyer, Julian Conrad, Pål Stenmark
The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a "closed" domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor...
June 23, 2017: EMBO Reports
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