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Cryo electron microscopy

Nazario Martin, Alicia López-Andarias, Javier López-Andarias, Carmen Atienza, Francisco J Chichón, José L Carrascosa
Supramolecular chemistry has evolved from the traditional focus on thermodynamic on-pathways to the complex study of kinetic off-pathways, which are strongly dependent of the environmental conditions. Moreover, the control over pathway complexity allows obtaining nanostructures that are inaccessible through spontaneous thermodynamic processes. Here, we present a family of peptide-based π-extended tetrathiafulvalene (exTTF) molecules which show two self-assembly pathways leading to two distinct J-aggregates, namely metastable (M) and thermodynamic (T), with different spectroscopic, chiroptical and electrochemical behavior...
March 14, 2018: Chemistry: a European Journal
Jonathan M Grimes, David R Hall, Alun W Ashton, Gwyndaf Evans, Robin L Owen, Armin Wagner, Katherine E McAuley, Frank von Delft, Allen M Orville, Thomas Sorensen, Martin A Walsh, Helen M Ginn, David I Stuart
Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments...
February 1, 2018: Acta Crystallographica. Section D, Structural Biology
Kevin Cowtan, Jon Agirre
Refinement is a critical step in the determination of a model which explains the crystallographic observations and thus best accounts for the missing phase components. The scattering density is usually described in terms of atomic parameters; however, in macromolecular crystallography the resolution of the data is generally insufficient to determine the values of these parameters for individual atoms. Stereochemical and geometric restraints are used to provide additional information, but produce interrelationships between parameters which slow convergence, resulting in longer refinement times...
February 1, 2018: Acta Crystallographica. Section D, Structural Biology
Oliver S Smart, Vladimír Horský, Swanand Gore, Radka Svobodová Vařeková, Veronika Bendová, Gerard J Kleywegt, Sameer Velankar
Realising the importance of assessing the quality of the biomolecular structures deposited in the Protein Data Bank (PDB), the Worldwide Protein Data Bank (wwPDB) partners established Validation Task Forces to obtain advice on the methods and standards to be used to validate structures determined by X-ray crystallography, nuclear magnetic resonance spectroscopy and three-dimensional electron cryo-microscopy. The resulting wwPDB validation pipeline is an integral part of the wwPDB OneDep deposition, biocuration and validation system...
March 1, 2018: Acta Crystallographica. Section D, Structural Biology
Roman I Koning, Abraham J Koster, Thomas H Sharp
Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware...
March 8, 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
M G Herrera, D S Vazquez, R Sreij, M Drechsler, Y Hertle, T Hellweg, V I Dodero
Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-...
February 27, 2018: Colloids and Surfaces. B, Biointerfaces
Katharina Braunger, Stefan Pfeffer, Shiteshu Shrimal, Reid Gilmore, Otto Berninghausen, Elisabet C Mandon, Thomas Becker, Friedrich Förster, Roland Beckmann
Protein synthesis, transport and N-glycosylation are coupled at the mammalian endoplasmic reticulum (ER) by complex formation between the ribosome, the Sec61 protein-conducting channel and the oligosaccharyltransferase (OST). Here, we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit revealed how STT3A is integrated into the OST and how STT3 paralog specificity for translocon-associated OST is achieved...
March 8, 2018: Science
David E Timm, Valorie Bowman, Russell Madsen, Charles Rauch
Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry...
2018: PloS One
Lin Chen, Jing He, Salim Sazzed, Rayshawn Walker
Cryo-electron microscopy (cryo-EM) is a structure determination method for large molecular complexes. As more and more atomic structures are determined using this technique, it is becoming possible to perform statistical characterization of side-chain conformations. Two data sets were involved to characterize block lengths for each of the 18 types of amino acids. One set contains 9131 structures resolved using X-ray crystallography from density maps with better than or equal to 1.5 Å resolutions, and the other contains 237 protein structures derived from cryo-EM density maps with 2-4 Å resolutions...
March 8, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Nate Yoder, Craig Yoshioka, Eric Gouaux
Acid-sensing ion channels (ASICs) are trimeric, proton-gated and sodium-selective members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized. Existing X-ray structures of ASIC1a describe the conformations of the open and desensitized states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive...
March 7, 2018: Nature
Timothy Grant, Alexis Rohou, Nikolaus Grigorieff
We have developed new open-source software called cis TEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cis TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction...
March 7, 2018: ELife
Takamichi Kamigaki, Yosiko Ito, Yuri Nishino, Atsuo Miyazawa
Casein micelles are present in bovine milk as colloidal particles with diameters of 20-600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution...
March 2, 2018: Microscopy
Paula K Okuro, Iris Tavernier, Mohd D Bin Sintang, Andre G Skirtach, António A Vicente, Koen Dewettinck, Rosiane L Cunha
In this study, the effect of lecithin (LEC) on the crystallization and gelation of fruit wax (FW) with sunflower oil was researched. A synergistic effect on the gel strength was observed at FW : LEC ratios of 75 : 25 and 50 : 50, compared to the corresponding single component formulations (100 : 0 and 0 : 100). Even below the critical gelling concentration (Cg ) of FW, the addition of lecithin enabled gel formation. Lecithin affected the thermal behavior of the structure by delaying both crystallization and gel formation...
March 6, 2018: Food & Function
Yuxi Liu, Shane Gonen, Tamir Gonen, Todd O Yeates
Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Daan Vorselen, Margherita Marchetti, Carmen López-Iglesias, Peter J Peters, Wouter H Roos, Gijs J L Wuite
Small multilamellar vesicles may have benefits over unilamellar vesicles for drug delivery, such as an increased volume for hydrophobic drugs. In addition, their altered mechanical properties might be beneficial for cellular uptake. Here, we show how atomic force microscopy (AFM) can be used to detect and characterize multilamellar vesicles. We quantify the size of each break event occurring during AFM nanoindentations, which shows good agreement with the thickness of supported lipid bilayers. Analyzing the size and number of these events for individual vesicles allows us to distinguish between vesicles consisting of 1 up to 5 bilayers...
March 5, 2018: Nanoscale
Philip Ketterer, Adithya N Ananth, Diederik S Laman Trip, Ankur Mishra, Eva Bertosin, Mahipal Ganji, Jaco van der Torre, Patrick Onck, Hendrik Dietz, Cees Dekker
The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant...
March 2, 2018: Nature Communications
Kevin W Huynh, Jiansen Jiang, Natalia Abuladze, Kirill Tsirulnikov, Liyo Kao, Xuesi Shao, Debra Newman, Rustam Azimov, Alexander Pushkin, Z Hong Zhou, Ira Kurtz
Na+ -coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+ -coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3...
March 2, 2018: Nature Communications
Xieyang Guo, Alexander G Myasnikov, James Chen, Corinne Crucifix, Gabor Papai, Maria Takacs, Patrick Schultz, Albert Weixlbaumer
Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel...
March 1, 2018: Molecular Cell
Max E Wilkinson, Pei-Chun Lin, Clemens Plaschka, Kiyoshi Nagai
The removal of noncoding introns from pre-messengerRNA(pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism...
March 1, 2018: Annual Review of Biophysics
Lorenzo I Finci, Xiaofeng Zhang, Xiuliang Huang, Qiang Zhou, Jennifer Tsai, Teng Teng, Anant Agrawal, Betty Chan, Sean Irwin, Craig Karr, Andrew Cook, Ping Zhu, Dominic Reynolds, Peter G Smith, Peter Fekkes, Silvia Buonamici, Nicholas A Larsen
Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design...
February 1, 2018: Genes & Development
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