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Peter S Shen, Xiaoyong Yang, Paul G DeCaen, Xiaowen Liu, David Bulkley, David E Clapham, Erhu Cao
The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction...
October 20, 2016: Cell
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
Zheng Liu, Cristina Gutierrez-Vargas, Jia Wei, Robert A Grassucci, Noel Espina, Susan Madison-Antenucci, Liang Tong, Joachim Frank
With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg(2+) and Zn(2+) ...
October 17, 2016: Protein Science: a Publication of the Protein Society
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
Sean Ekins, John Liebler, Bruno J Neves, Warren G Lewis, Megan Coffee, Rachelle Bienstock, Christopher Southan, Carolina H Andrade
The Zika virus (ZIKV) is a flavivirus of the family Flaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it either in vitro or in vivo...
2016: F1000Research
Tian-Min Fu, Yang Li, Alvin Lu, Zongli Li, Parimala R Vajjhala, Anthony C Cruz, Devendra B Srivastava, Frank DiMaio, Pawel A Penczek, Richard M Siegel, Katryn J Stacey, Edward H Egelman, Hao Wu
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells...
October 20, 2016: Molecular Cell
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
Joanna Rorbach, Fei Gao, Christopher A Powell, Aaron D'Souza, Robert N Lightowlers, Michal Minczuk, Zofia M Chrzanowska-Lightowlers
The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNA(Val) compared with the porcine use of mt-tRNA(Phe) We have explored this observation further...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
Anne Houdusse, H Lee Sweeney
How myosin interacts with actin to generate force is a subject of considerable controversy. The major debate centers on understanding at what point in force generation the inorganic phosphate is released with respect to the lever arm swing, or powerstroke. Resolving the controversy is essential for understanding how force is produced as well as the mechanisms underlying disease-causing mutations in myosin. Recent structural insights into the powerstroke have come from a high-resolution structure of myosin in a previously unseen state and from an electron cryomicroscopy (cryo-EM) 3D reconstruction of the actin-myosin-MgADP complex...
October 4, 2016: Trends in Biochemical Sciences
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
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
Ping Zhu, Guohong Li
Genomic DNA is hierarchically packaged into chromatin in eukaryotes. As a central-level chromatin structure between nucleosomal arrays and higher order organizations, 30 nm chromatin fiber, and its dynamics play a crucial role in regulating DNA accessibility for gene transcription. However, despite extensive efforts over three decades, the higher-order structure of the 30 nm chromatin fiber remains unresolved and controversial. We have recently reconstituted the 30 nm chromatin fibers from 12 nucleosomal arrays in vitro in the presence of linker histone H1, and determined their cryo-EM structures at resolution of 11 Å (Song et al...
October 5, 2016: IUBMB Life
Zhongjun Hu, Dianne W Taylor, Michael K Reedy, Robert J Edwards, Kenneth A Taylor
We describe a cryo-electron microscopy three-dimensional image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species...
September 2016: Science Advances
Anna Raffaello, Cristina Mammucari, Gaia Gherardi, Rosario Rizzuto
In recent years, rapid discoveries have been made relating to Ca(2+) handling at specific organelles that have important implications for whole-cell Ca(2+) homeostasis. In particular, the structures of the endoplasmic reticulum (ER) Ca(2+) channels revealed by electron cryomicroscopy (cryo-EM), continuous updates on the structure, regulation, and role of the mitochondrial calcium uniporter (MCU) complex, and the analysis of lysosomal Ca(2+) signaling are milestones on the route towards a deeper comprehension of the complexity of global Ca(2+) signaling...
September 28, 2016: Trends in Biochemical Sciences
Eva Nogales, Robert K Louder, Yuan He
Single particle cryo-Electron Microscopy (cryo-EM) is a technique that allows the structural characterization of macromolecules without the need for crystallization. For certain type of samples that are ideally suited for cryo-EM studies it has been possible to reach high-resolution structures following relatively standard procedures. Other biological systems remain highly challenging, even for cryo-EM. Challenges may involve the scarcity of the sample, poor stability of the complexes, and most often, the intrinsic flexibility of biological molecules...
September 30, 2016: Current Opinion in Structural Biology
Matthew G Iadanza, Anna J Higgins, Bob Schiffrin, Antonio N Calabrese, David J Brockwell, Alison E Ashcroft, Sheena E Radford, Neil A Ranson
The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a 'lateral gating' motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding...
2016: Nature Communications
Tanmay A M Bharat, Sjors H W Scheres
Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis...
November 2016: Nature Protocols
M Hunter Giese, Alison Gardner, Angela Hansen, Michael C Sanguinetti
Large conductance K(+) -selective Slo2 channels are in a closed state unless activated by elevated [Na(+) ]i . Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. Here we evaluate two other potential mechanisms for stabilization of Slo2 channels in a closed state: 1) dewetting and collapse of the inner pore (hydrophobic gating), and 2) constriction of the inner pore by tight criss-crossing of the cytoplasmic ends of the S6 α-helical segments...
September 28, 2016: Journal of Physiology
Yoshimasa Takizawa, Elad Binshtein, Amanda L Erwin, Tasia M Pyburn, Kathleen F Mittendorf, Melanie D Ohi
Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations...
October 6, 2016: Protein Science: a Publication of the Protein Society
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