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 Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles

2020 ◽  
Vol 6 (30) ◽  
pp. eaba8381 ◽  
Author(s):  
Oleksiy Kovtun ◽  
Veronica Kane Dickson ◽  
Bernard T. Kelly ◽  
David J. Owen ◽  
John A. G. Briggs
Keyword(s):  
Electron Microscopy ◽  
Adaptor Protein ◽  
Protein Composition ◽  
Binding Mode ◽  
Coated Vesicles ◽  
Conformational Rearrangement ◽  
Multiple Interfaces ◽  
Cryo Electron Microscopy ◽  
Subtomogram Averaging ◽  
Key Steps

Clathrin-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. Adaptor protein complex (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 (phosphatidylinositol 4,5-bisphosphate)–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 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 Template-free 13-protofilament microtubule–MAP assembly visualized at 8 Å resolution

2010 ◽  
Vol 191 (3) ◽  
pp. 463-470 ◽  
Author(s):  
Franck J. Fourniol ◽  
Charles V. Sindelar ◽  
Béatrice Amigues ◽  
Daniel K. Clare ◽  
Geraint Thomas ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Information ◽  
Binding Mode ◽  
Microtubule Associated Proteins ◽  
Cryo Electron Microscopy ◽  
Associated Proteins ◽  
Template Free ◽  
Map Function ◽  
Insight Into

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

Ultrastructural aspects of coated vesicles in tobacco protoplasts

1981 ◽  
Vol 59 (7) ◽  
pp. 1307-1313 ◽  
Author(s):  
P. van der Valk ◽  
L. C. Fowke
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Plasma Membrane ◽  
Coated Vesicles ◽  
Thin Sections ◽  
Tobacco Protoplasts ◽  
Vesicle Membranes ◽  
Transmission Electron ◽  
Large Numbers ◽  
Inner Surface

The ultrastructure and distribution of coated vesicles in isolated tobacco protoplasts were investigated using transmission electron microscopy of thin sections of whole protoplasts and stained plasma membrane preparations obtained by osmotic bursting of protoplasts attached to coated microscope grids. Large numbers of coated vesicles were associated with both the plasma membrane and the maturing face of dictyosomes. Dictyosome associated coated vesicles were smaller and had less distinct coats and vesicle membranes than those associated with the plasma membrane. Honeycomb structures believed to be aggregations of coats were also associated with the inner surface of the plasma membrane. Our data suggest that coated vesicles are produced by the Golgi apparatus, fuse with the plasma membrane, their coats remaining attached, at least temporarily, to the plasma membrane inner surface.

scholarly journals Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS

2018 ◽  
Author(s):  
Kangkang Song ◽  
Zhiguo Shang ◽  
Xiaofeng Fu ◽  
Xiaochu Lou ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hybrid Method ◽  
Atomic Resolution ◽  
Signal Loss ◽  
Nanometer Resolution ◽  
Cryo Electron Microscopy ◽  
Microscopy Method ◽  
Subtomogram Averaging ◽  
Electron Microscopy Method ◽  
Crowded Environments

AbstractThe resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microcopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We developed a novel hybrid-method called “TomographY-Guided 3D REconstruction of Subcellular Structures” (TYGRESS) that combines cryo-ET with SP-cryo-EM to achieve close-to-nanometer resolution of complexes inside crowded environments. Using TYGRESS, we determined the native 3D structures of the intact ciliary axoneme with up to 12 Å resolution. These results reveal many structures and details that were not visible by cryo-ET. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging, bringing us a step closer to (pseudo-)atomic models of cells.One Sentence SummaryA hybrid cryo-electron microscopy method reveals subcellular structures at unprecedented resolution.

scholarly journals John Briggs: A closer look at HIV and coated vesicles

2013 ◽  
Vol 201 (7) ◽  
pp. 966-967
Author(s):  
Caitlin Sedwick
Keyword(s):  
Electron Microscopy ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Cryo Electron Microscopy

Briggs studies the organization of viral and cellular coat proteins using cryo-electron microscopy.

scholarly journals Combining high throughput and high quality for cryo-electron microscopy data collection

2020 ◽  
Vol 76 (8) ◽  
pp. 724-728
Author(s):  
Felix Weis ◽  
Wim J. H. Hagen
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Electron Tomography ◽  
3D Structure ◽  
Particle Analysis ◽  
High Resolution Data ◽  
Cryo Electron Microscopy ◽  
Microscopy Data ◽  
Subtomogram Averaging

Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.

scholarly journals User Manual for Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS)

2019 ◽  
Author(s):  
Zhiguo Shang ◽  
Kangkang Song ◽  
Xiaofeng Fu ◽  
Xiaochu Lou ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Intact Cells ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Crowded Environment

Abstract Recent advances in cryo-electron microscopy (cryo-EM) are paving the way to determining isolated three-dimensional (3D) macromolecular structures at near-atomic resolution using single-particle cryo-electron microscopy (SP-cryo-EM). However, determining the subcellular structures in intact cells and organelles using cryo-electron tomography (cryo-ET) and subtomogram averaging, another cryo-EM technique, with comparable resolution remains a challenge. Current methodologies can only reach a resolution of several nanometers in most samples studied. Here, we introduce a new hybrid method, called Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS) that is able to achieve structural determination of subcellular structures within their natural crowded environment with nanometer-resolution by combining the advantages of cryo-ET and SP-cryo-EM.

scholarly journals Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Lili Wu ◽  
Qian Chen ◽  
Kefang Liu ◽  
Jia Wang ◽  
Pengcheng Han ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Intermediate Host ◽  
Molecular Basis ◽  
Binding Mode ◽  
Domestic Animals ◽  
Broad Host Range ◽  
Receptor Binding Domain ◽  
Wild Animals ◽  
Cryo Electron Microscopy ◽  
Shed Light

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.

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