scholarly journals Molecular mechanism of the Orai channel activation

2018 ◽  
Author(s):  
Xiaofen Liu ◽  
Guangyan Wu ◽  
Yi Yu ◽  
Xiaozhe Chen ◽  
Renci Ji ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Plasma Membrane ◽  
Potential Gradient ◽  
State Structure ◽  
Channel Activation ◽  
Cryo Electron Microscopy ◽  
Cytosolic Side ◽  
Ion Conducting ◽  
Channel Assembly

The Orai channel is characterized by voltage independence, low conductance and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane. How the channel is activated and promotes Ca2+ permeation are not well understood. Here, we report the crystal structure and cryo-electron microscopy reconstruction of a Drosophila melanogaster Orai mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which six TM1 helices in the center form the ion-conducting pore, and six TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation and Ca2+ permeation.

scholarly journals Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures of Enterococcus gallinarum and Streptococcus gordonii Septa Revealed by Cryo-Electron Microscopy of Vitreous Sections

2006 ◽  
Vol 188 (18) ◽  
pp. 6652-6660 ◽  
Author(s):  
Benoît Zuber ◽  
Marisa Haenni ◽  
Tânia Ribeiro ◽  
Kathrin Minnig ◽  
Fátima Lopes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Granular Layer ◽  
Streptococcus Gordonii ◽  
Periplasmic Space ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cryo Electron Microscopy ◽  
Enterococcus Gallinarum ◽  
Peptidoglycan Layer

ABSTRACT High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

scholarly journals Architecture of the human PI4KIIIα lipid kinase complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13720-13725 ◽  
Author(s):  
Joshua A. Lees ◽  
Yixiao Zhang ◽  
Michael S. Oh ◽  
Curtis M. Schauder ◽  
Xiaoling Yu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Active Sites ◽  
Structural Complexity ◽  
Signaling Molecules ◽  
Cell Physiology ◽  
Lipid Kinase ◽  
Membrane Interaction ◽  
Regulatory Subunits ◽  
Cryo Electron Microscopy

Plasma membrane (PM) phosphoinositides play essential roles in cell physiology, serving as both markers of membrane identity and signaling molecules central to the cell’s interaction with its environment. The first step in PM phosphoinositide synthesis is the conversion of phosphatidylinositol (PI) to PI4P, the precursor of PI(4,5)P2 and PI(3,4,5)P3. This conversion is catalyzed by the PI4KIIIα complex, comprising a lipid kinase, PI4KIIIα, and two regulatory subunits, TTC7 and FAM126. We here report the structure of this complex at 3.6-Å resolution, determined by cryo-electron microscopy. The proteins form an obligate ∼700-kDa superassembly with a broad surface suitable for membrane interaction, toward which the kinase active sites are oriented. The structural complexity of the assembly highlights PI4P synthesis as a major regulatory junction in PM phosphoinositide homeostasis. Our studies provide a framework for further exploring the mechanisms underlying PM phosphoinositide regulation.

scholarly journals Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

2020 ◽  
Author(s):  
Oleksiy Kovtun ◽  
Veronica Kane Dickson ◽  
Bernard T. Kelly ◽  
David. J. Owen ◽  
John A. G. Briggs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Protein Composition ◽  
Binding Mode ◽  
Coated Vesicles ◽  
Conformational Rearrangement ◽  
Multiple Interfaces ◽  
Cryo Electron Microscopy ◽  
Subtomogram Averaging ◽  
Key Steps

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

scholarly journals Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Herve Celia ◽  
Istvan Botos ◽  
Xiaodan Ni ◽  
Tara Fox ◽  
Natalia De Val ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
High Resolution ◽  
Outer Membrane ◽  
Motor Function ◽  
Structural Information ◽  
Molecular Motor ◽  
Single Copy ◽  
Proton Motive Force ◽  
Cryo Electron Microscopy

Abstract The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

scholarly journals Structural insights into stressosome assembly

IUCrJ ◽  
2019 ◽  
Vol 6 (5) ◽  
pp. 938-947 ◽  
Author(s):  
Eunju Kwon ◽  
Deepak Pathak ◽  
Han-ul Kim ◽  
Pawan Dahal ◽  
Sung Chul Ha ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Environmental Stress ◽  
Assembly Model ◽  
Bacterial Viability ◽  
Binding Interface ◽  
Cryo Electron Microscopy ◽  
Stress Signals ◽  
Structural Insights

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA–RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA–RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

scholarly journals Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Doudou Wang ◽  
Ryo Nitta ◽  
Manatsu Morikawa ◽  
Hiroaki Yajima ◽  
Shigeyuki Inoue ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Dual Function ◽  
Microtubule Depolymerization ◽  
Multiple Strategies ◽  
Cryo Electron Microscopy ◽  
Cilium Length ◽  
Dual Functions

The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis.

scholarly journals Interaction of Decay-Accelerating Factor with Echovirus 7

2010 ◽  
Vol 84 (24) ◽  
pp. 12665-12674 ◽  
Author(s):  
Pavel Plevka ◽  
Susan Hafenstein ◽  
Katherine G. Harris ◽  
Javier O. Cifuente ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Binding Site ◽  
Symmetry Axis ◽  
Cellular Receptor ◽  
Icosahedral Symmetry ◽  
Decay Accelerating Factor ◽  
Cryo Electron Microscopy ◽  
The Family ◽  
Initial Process

ABSTRACT Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.

Electron microscopy of endocardial cell populations

1978 ◽  
Vol 36 (2) ◽  
pp. 486-487
Author(s):  
T. G. Sarphie ◽  
C. R. Comer ◽  
D. J. Allen
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cellular Transformation ◽  
Cell Populations ◽  
Cell Surfaces ◽  
Kb Cells ◽  
Dna Viruses ◽  
Cell Cycles ◽  
Ultrastructural Studies ◽  
Endocardial Cell

Previous ultrastructural studies have characterized surface morphology during norma cell cycles in an attempt to associate specific changes with specific metabolic processes occurring within the cell. It is now known that during the synthetic ("S") stage of the cycle, when DNA and other nuclear components are synthesized, a cel undergoes a doubling in volume that is accompanied by an increase in surface area whereby its plasma membrane is elaborated into a variety of processes originally referred to as microvilli. In addition, changes in the normal distribution of glycoproteins and polysaccharides derived from cell surfaces have been reported as depreciating after cellular transformation by RNA or DNA viruses and have been associated with the state of growth, irregardless of the rate of proliferation. More specifically, examination of the surface carbohydrate content of synchronous KB cells were shown to be markedly reduced as the cell population approached division Comparison of hamster kidney fibroblasts inhibited by vinblastin sulfate while in metaphase with those not in metaphase demonstrated an appreciable decrease in surface carbohydrate in the former.

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