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Cryo electron microscopy

William A Cantara, Erik D Olson, Karin Musier-Forsyth
In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist...
October 21, 2016: Methods: a Companion to Methods in Enzymology
Tofayel Ahmed, Zhan Yin, Shashi Bhushan
Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit...
October 20, 2016: Scientific Reports
Daniel R Ripoll, Ilja Khavrutskii, Anders Wallqvist, Sidhartha Chaudhury
Cryo-electron-microscopy (cryo-EM) structures of flaviviruses reveal significant variation in epitope occupancy across different monoclonal antibodies that have largely been attributed to epitope-level differences in conformation or accessibility that affect antibody binding. The consequences of these variations for macroscopic properties such as antibody binding and neutralization are the results of the law of mass action-a stochastic process of innumerable binding and unbinding events between antibodies and the multiple binding sites on the flavivirus in equilibrium-that cannot be directly imputed from structure alone...
October 18, 2016: Biophysical Journal
Emil F Khisamutdinov, Daniel L Jasinski, Hui Li, Kaiming Zhang, Wah Chiu, Peixuan Guo
Constructing containers with defined shape and size to load and protect therapeutics and subsequently control their release in the human body has long been a dream. The fabrication of 3D RNA prisms, characterized by atomic force microscopy, cryo-electron microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis, is reported for the loading and protection of small molecules, proteins, small RNA molecules, and their controlled release.
October 19, 2016: Advanced Materials
Anna Mularski, Jonathan Wilksch, Eric Hanssen, Jian Li, Takehiro Tomita, Sacha James Pidot, Tim Stinear, Frances Separovic, Dick Strugnell
Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1...
October 17, 2016: European Biophysics Journal: EBJ
Christopher J Benjamin, Kyle J Wright, Scott C Bolton, Seok-Hee Hyun, Kyle Krynski, Mahima Grover, Guimei Yu, Fei Guo, Tamara L Kinzer-Ursem, Wen Jiang, David H Thompson
We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N(α), N(α)-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E...
October 17, 2016: Scientific Reports
Huan-Yuan Chen, Dapi Meng-Lin Chiang, Zi-Jing Lin, Chia-Chun Hsieh, Gung-Chian Yin, I-Chun Weng, Peter Guttermann, Stephan Werner, Katja Henzler, Gerd Schneider, Lee-Jene Lai, Fu-Tong Liu
Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation...
October 17, 2016: Scientific Reports
Yong-Jiang Zhang, Fulton E Rockwell, Adam C Graham, Teressa Alexander, N Michele Holbrook
We report a novel form of xylem dysfunction in angiosperms: reversible collapse of the xylem conduits of the smallest vein orders that demarcate and intrusively irrigate the areoles of Quercus rubra leaves. Cryo-scanning electron microscopy revealed gradual increases in collapse from ~ -2 MPa down to ~ -3 MPa, saturating thereafter (to -4 MPa). Over this range cavitation remained negligible in these veins. Imaging of rehydration experiments showed spatially variable recovery from collapse within 20 seconds, and complete recovery after two minutes...
October 12, 2016: Plant Physiology
Igor Orlov, Alexander G Myasnikov, Leonid Andronov, S Kundhavai Natchiar, Heena Khatter, Brice Beinsteiner, Jean-François Ménétret, Isabelle Hazemann, Kareem Mohideen, Karima Tazibt, Rachel Tabaroni, Hanna Kratzat, Nadia Djabeur, Tatiana Bruxelles, Finaritra Raivoniaina, Lorenza di Pompeo, Morgan Torchy, Isabelle Billas, Alexandre Urzhumtsev, Bruno P Klaholz
After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This "revolution in resolution" is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on CMOS devices...
October 12, 2016: Biology of the Cell
Andrea D Merg, Jennifer C Boatz, Abhishek Mandal, Gongpu Zhao, Soumitra Mokashi-Punekar, Chong Liu, Xianting Wang, Peijun Zhang, Patrick C A van der Wel, Nathaniel L Rosi
Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAu(M-ox))2 (PEPAu(M-ox) = AYSSGAPPM(ox)PPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles...
October 11, 2016: Journal of the American Chemical Society
Yaser Kashcooli, Keunhan Park, Arijit Bose, Michael Lewis Greenfield, Geoffrey D Bothun
Layer-by-layer deposition of polyelectrolytes (PEs) onto self-assembled liposomes represents an alternative to PE deposition on solid particles for the formation of hollow nanoscale capsules. This work examines how competition between PE-liposome and inter-PE interactions drives the structure and colloidal stability of layersomes. Unlike solid particles, liposomes respond to adsorbed material through lipid reorganization and changes in size and shape. This responsive nature could yield new types of layered PE structures...
October 10, 2016: Biomacromolecules
Yi Liu, Nathalie Claes, Bastian Trepka, Sara Bals, Peter R Lang
In this article we report on the synthesis and characterization of a system of colloidal spheres suspended in an aqueous solvent which can be refractive index-matched, thus allowing for investigations of the particle near-wall dynamics by evanescent wave dynamic light scattering at concentrations up to the isotropic to ordered transition and beyond. The particles are synthesized by copolymerization of a fluorinated acrylic ester monomer with a polyethylene-glycol (PEG) oligomer by surfactant free emulsion polymerization...
September 5, 2016: Soft Matter
Idlir Liko, Timothy M Allison, Jonathan Ts Hopper, Carol V Robinson
With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes...
October 6, 2016: Current Opinion in Structural Biology
Nicolas Coudray, Sean L Seyler, Ralph Lasala, Zhening Zhang, Kathy M Clark, Mark E Dumont, Alexis Rohou, Oliver Beckstein, David L Stokes
Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization...
October 7, 2016: Protein Science: a Publication of the Protein Society
Leïla Zerkoune, Sylviane Lesieur, Jean-Luc Putaux, Luc Choisnard, Annabelle Gèze, Denis Wouessidjewe, Borislav Angelov, Corinne Vebert-Nardin, James Doutch, Angelina Angelova
Soft mesoporous hierarchically structured particles were created by the self-assembly of an amphiphilic deep cavitand cyclodextrin βCD-nC10 (degree of substitution n = 7.3), with a nanocavity grafted by multiple alkyl (C10) chains on the secondary face of the βCD macrocycle through enzymatic biotransesterification, and the nonlamellar lipid monoolein (MO). The effect of the non-ionic dispersing agent polysorbate 80 (P80) on the liquid crystalline organization of the nanocarriers and their stability was studied in the context of vesicle-to-cubosome transition...
September 13, 2016: Soft Matter
R J G Hulswit, C A M de Haan, B-J Bosch
Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-CoV. Due to their inherent genetic variability, it is inevitable that new cross-species transmission events of these enveloped, positive-stranded RNA viruses will occur. Research into these medical and veterinary important pathogens-sparked by the SARS and MERS outbreaks-revealed important principles of inter- and intraspecies tropism changes...
2016: Advances in Virus Research
Jan Huebinger, Hong-Mei Han, Markus Grabenbauer
Rapid cooling of aqueous solutions is a useful approach for two important biological applications: (I) cryopreservation of cells and tissues for long-term storage, and (II) cryofixation for ultrastructural investigations by electron and cryo-electron microscopy. Usually, both approaches are very different in methodology. Here we show that a novel, fast and easy to use cryofixation technique called self-pressurized rapid freezing (SPRF) is-after some adaptations-also a useful and versatile technique for cryopreservation...
2016: PloS One
Guray Kuzu, Ozlem Keskin, Ruth Nussinov, Attila Gursoy
The structures of protein assemblies are important for elucidating cellular processes at the molecular level. Three-dimensional electron microscopy (3DEM) is a powerful method to identify the structures of assemblies, especially those that are challenging to study by crystallography. Here, a new approach, PRISM-EM, is reported to computationally generate plausible structural models using a procedure that combines crystallographic structures and density maps obtained from 3DEM. The predictions are validated against seven available structurally different crystallographic complexes...
October 1, 2016: Acta Crystallographica. Section D, Structural Biology
Jin Seob Kim, Bijan Afsari, Gregory S Chirikjian
Cryo-electron microscopy (EM) and small angle X-ray scattering (SAXS) are two different data acquisition modalities often used to glean information about the structure of large biomolecular complexes in their native states. A SAXS experiment is generally considered fast and easy but unveils the structure at very low resolution, whereas a cryo-EM experiment needs more extensive preparation and postacquisition computation to yield a three-dimensional (3D) density map at higher resolution. In certain applications, we may need to verify whether the data acquired in the SAXS and cryo-EM experiments correspond to the same structure (e...
October 6, 2016: Journal of Computational Biology: a Journal of Computational Molecular Cell Biology
Qingxia Zhong, Anna Carratala, Sergey Nazarov, Ricardo C Guerrero-Ferreira, Laura Piccinini, Virginie Bachmann, Petr G Leiman, Tamar Kohn
Common water disinfectants like chlorine have been reported to select for resistant viruses, yet little attention has been devoted to characterizing disinfection resistance. Here, we investigated the resistance of MS2 coliphage to inactivation by chlorine dioxide (ClO2). ClO2 inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. ClO2-resistant virus populations emerged after repeated cycles of ClO2 disinfection followed by regrowth, but also after dilution-regrowth cycles in the absence of ClO2...
October 6, 2016: Environmental Science & Technology
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