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https://www.readbyqxmd.com/read/28340353/molecular-structure-of-the-human-cftr-ion-channel
#1
Fangyu Liu, Zhe Zhang, László Csanády, David C Gadsby, Jue Chen
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening...
March 23, 2017: Cell
https://www.readbyqxmd.com/read/28340349/structure-reveals-mechanisms-of-viral-suppressors-that-intercept-a-crispr-rna-guided-surveillance-complex
#2
Saikat Chowdhury, Joshua Carter, MaryClare F Rollins, Sarah M Golden, Ryan N Jackson, Connor Hoffmann, Lyn'Al Nosaka, Joseph Bondy-Denomy, Karen L Maxwell, Alan R Davidson, Elizabeth R Fischer, Gabriel C Lander, Blake Wiedenheft
Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3...
March 23, 2017: Cell
https://www.readbyqxmd.com/read/28340337/structural-basis-of-rna-polymerase-i-transcription-initiation
#3
Christoph Engel, Tobias Gubbey, Simon Neyer, Sarah Sainsbury, Christiane Oberthuer, Carlo Baejen, Carrie Bernecky, Patrick Cramer
Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis...
March 23, 2017: Cell
https://www.readbyqxmd.com/read/28340334/advances-in-phase-plate-cryo-em-imaging-of-dna-and-nucleosomes
#4
Eugene Y D Chua, Sara Sandin
Contrast in electron cryo-microscopy (cryo-EM) is limited by the weak phase and radiation sensitive nature of biologic samples embedded in vitrified ice. We have recently shown that a new contrast enhancement technique utilizing the Volta phase plate can be combined with single particle analysis to determine the structure of a small chromatin complex, the nucleosome core particle, at near-atomic resolution. Here, we discuss advantages and limitations of the technique in terms of data collection, particle detection, and visualization of individual DNA molecules and higher-order chromatin structure...
February 7, 2017: Nucleus
https://www.readbyqxmd.com/read/28325834/structural-model-of-dodecameric-heat-shock-protein-hsp21-flexible-n-terminal-arms-interact-with-client-proteins-while-c-terminal-tails-maintain-the-dodecamer-and-chaperone-activity
#5
Gudrun Rutsdottir, Johan Härmark, Yoran Weide, Hans Hebert, Morten Ib Rasmussen, Sven Wernersson, Michal Respondek, Mikael Akke, Peter Højrup, Philip J B Koeck, Christopher A G Söderberg, Cecilia Emanuelsson
Small heat shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently there is only one high-resolution structure of a plant sHsp published, of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminals arms than Hsp16.9, with a functionally important and conserved methionine-rich motif...
March 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28315749/integrated-structural-biology-to-unravel-molecular-mechanisms-of-protein-rna-recognition
#6
REVIEW
Andreas Schlundt, Jan-Niklas Tants, Michael Sattler
Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation...
March 15, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28300532/mechanism-of-ribosome-rescue-by-arfa-and-rf2
#7
Gabriel Demo, Egor Svidritskiy, Rohini Madireddy, Ruben Diaz-Avalos, Timothy Grant, Nikolaus Grigorieff, Duncan Sousa, Andrei A Korostelev
ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center...
March 16, 2017: ELife
https://www.readbyqxmd.com/read/28286002/a-fast-and-effective-microfluidic-spraying-plunging-method-for-high-resolution-single-particle-cryo-em
#8
Xiangsong Feng, Ziao Fu, Sandip Kaledhonkar, Yuan Jia, Binita Shah, Amy Jin, Zheng Liu, Ming Sun, Bo Chen, Robert A Grassucci, Yukun Ren, Hongyuan Jiang, Joachim Frank, Qiao Lin
We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms...
March 6, 2017: Structure
https://www.readbyqxmd.com/read/28283067/transporters-revealed
#9
Jyh-Yeuan Lee, Daniel M Rosenbaum
The subfamily C ATP-binding cassette (ABCC) transporters mediate multidrug resistance and ion conductance regulation. Recent atomic or near-atomic resolution structures of three physiologically significant ABCC transporters (MRP1, SUR1, and CFTR), determined by using single-particle cryo-electron microscopy (cryo-EM), reveal structural details that help explain the wide functional diversity of this ABC transporter subfamily.
March 9, 2017: Cell
https://www.readbyqxmd.com/read/28281548/structure-and-assembly-of-scalable-porous-protein-cages
#10
Eita Sasaki, Daniel Böhringer, Michiel van de Waterbeemd, Marc Leibundgut, Reinhard Zschoche, Albert J R Heck, Nenad Ban, Donald Hilvert
Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively...
March 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28274636/enhancing-enterovirus-a71-vaccine-production-yield-by-microcarrier-profusion-bioreactor-culture
#11
Chia-Chyi Liu, Suh-Chin Wu, Shang-Rung Wu, Hsiao-Yu Lin, Meng-Shin Guo, Alan Yung-Chih Hu, Yen-Hung Chow, Jen-Ron Chiang, Dar-Bin Shieh, Pele Chong
Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated...
March 6, 2017: Vaccine
https://www.readbyqxmd.com/read/28271489/the-pyruvate-dehydrogenase-complex-and-related-assemblies-in-health-and-disease
#12
Olwyn Byron, John Gordon Lindsay
The family of 2-oxoacid dehydrogenase complexes (2-OADC), typified by the pyruvate dehydrogenase multi-enzyme complex (PDC) as its most prominent member, are massive molecular machines (Mr, 4-10 million) controlling key steps in glucose homeostasis (PDC), citric acid cycle flux (OGDC, 2-oxoglutarate dehydrogenase) and the metabolism of the branched-chain amino acids, leucine, isoleucine and valine (BCOADC, branched-chain 2-OADC). These highly organised mitochondrial arrays, composed of multiple copies of three separate enzymes, have been widely studied as paradigms for the analysis of enzyme cooperativity, substrate channelling, protein-protein interactions and the regulation of activity by phosphorylation ...
2017: Sub-cellular Biochemistry
https://www.readbyqxmd.com/read/28271488/the-aminoacyl-trna-synthetase-complex
#13
Marc Mirande
Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes...
2017: Sub-cellular Biochemistry
https://www.readbyqxmd.com/read/28271471/structure-and-function-of-the-stressosome-signalling-hub
#14
Jan Pané-Farré, Maureen B Quin, Richard J Lewis, Jon Marles-Wright
The stressosome is a multi-protein signal integration and transduction hub found in a wide range of bacterial species. The role that the stressosome plays in regulating the transcription of genes involved in the general stress response has been studied most extensively in the Gram-positive model organism Bacillus subtilis. The stressosome receives and relays the signal(s) that initiate a complex phosphorylation-dependent partner switching cascade, resulting in the activation of the alternative sigma factor σ(B)...
2017: Sub-cellular Biochemistry
https://www.readbyqxmd.com/read/28270620/accurate-model-annotation-of-a-near-atomic-resolution-cryo-em-map
#15
Corey F Hryc, Dong-Hua Chen, Pavel V Afonine, Joanita Jakana, Zhao Wang, Cameron Haase-Pettingell, Wen Jiang, Paul D Adams, Jonathan A King, Michael F Schmid, Wah Chiu
Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata...
March 7, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28270563/the-sleeping-beauty-kissed-awake-new-methods-in-electron-microscopy-to-study-cellular-membranes
#16
REVIEW
Petr Chlanda, Jacomine Krijnse Locker
Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM...
March 7, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28269599/simultaneous-reconstruction-and-segmentation-with-the-mumford-shah-functional-for-electron-tomography
#17
Li Shen, Eric Todd Quinto, Shiqiang Wang, Ming Jiang
Electron micrography (EM) is an important method for determining the three-dimensional (3D) structure of macromolecular complexes and biological specimens. But there are several limitations such as poor signal-to-noise, limitation on range of tilt angles and sub-region subject to electron exposure, unintentional movements of the specimen, with EM systems that make the reconstruction procedure a severely ill-posed problem. A different choice of reconstruction method may lead to different results and create different additional artifacts in reconstructed images...
August 2016: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/28267408/insights-into-remodeling-events-during-eukaryotic-large-ribosomal-subunit-assembly-provided-by-high-resolution-cryo-em-structures
#18
Stephanie Biedka, Shan Wu, Amber J LaPeruta, Ning Gao, John L Woolford
Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction...
March 7, 2017: RNA Biology
https://www.readbyqxmd.com/read/28263324/cryo-em-structure-of-a-metazoan-separase-securin-complex-at-near-atomic-resolution
#19
Andreas Boland, Thomas G Martin, Ziguo Zhang, Jing Yang, Xiao-Chen Bai, Leifu Chang, Sjors H W Scheres, David Barford
Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase-securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain...
March 6, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28262565/effects-of-radiation-damage-in-studies-of-protein-dna-complexes-by-cryo-em
#20
REVIEW
M Mishyna, O Volokh, Ya Danilova, N Gerasimova, E Pechnikova, O S Sokolova
Nucleic acids are responsible for the storage, transfer and realization of genetic information in the cell, which provides correct development and functioning of organisms. DNA interaction with ligands ensures the safety of this information. Over the past 10 years, advances in electron microscopy and image processing allowed to obtain the structures of key DNA-protein complexes with resolution below 4Å. However, radiation damage is a limiting factor to the potentially attainable resolution in cryo-EM. The prospect and limitations of studying protein-DNA complex interactions using cryo-electron microscopy are discussed here...
February 21, 2017: Micron: the International Research and Review Journal for Microscopy
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