scholarly journals Nanometer resolution in situ structure of SARS-CoV-2 post-fusion spike

2021 ◽  
Author(s):  
Yun Zhu ◽  
Fei Sun ◽  
Xiangxi Wang ◽  
Linhua Tai ◽  
Guoliang Zhu ◽  
...  
Keyword(s):  
Pore Formation ◽  
Transmembrane Domain ◽  
Structural Information ◽  
Protein S ◽  
Fusion Pore ◽  
Electron Microscopic ◽  
Nanometer Resolution ◽  
Entry Mechanism ◽  
Resolution Structure

Abstract The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

scholarly journals Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein

2021 ◽  
Vol 118 (48) ◽  
pp. e2112703118
Author(s):  
Linhua Tai ◽  
Guoliang Zhu ◽  
Minnan Yang ◽  
Lei Cao ◽  
Xiaorui Xing ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Structural Information ◽  
Electron Tomography ◽  
Host Cells ◽  
Spike Protein ◽  
Fusion Pore ◽  
Nanometer Resolution ◽  
Glycosylation Sites ◽  
Entry Mechanism

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in β-propiolactone–inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.

scholarly journals Synaptobrevin-2 C-terminal Flexible Region Regulates the Discharge of Catecholamine through the Fusion Pore

2018 ◽  
Author(s):  
Annita N. Weiss
Keyword(s):  
Membrane Potential ◽  
Lipid Bilayer ◽  
Chromaffin Cells ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Flash Photolysis ◽  
Fusion Pore ◽  
Snare Protein ◽  
Head Groups ◽  
Flexible Region

AbstractThe discharge of neurotransmitters from vesicles is a regulated process. Synaptobrevin-2 a SNARE protein, participates in this process through its interaction with other SNARE and associate proteins. Synaptobrevin-2 transmembrane domain is embedded into the vesicle lipid bilayer except for its last three residues. These residues are hydrophilic and constitute synaptobrevin-2 C-terminal flexible region. This region interacts with the intravesicular lipid bilayer phosphate head groups to initiate the fusion pore formation. Here it is shown that, this region also modulates the intravesicular membrane potential thereby the discharged of catecholamine. Synapotobrevin-2 Y113 residue was mutated to lysine or glutamate. The effects of these mutations on the exocytotic process in chromaffin cells were assessed using capacitance measurements, combined with amperometry and stimulation by flash photolysis of caged Ca2+. Both Y113E and Y113K mutations reduced the amplitudes of vesicle fusions and reduced the rates of release of catecholamine molecules in quanta release events. Further investigation revealed that the proximity of these charged residues near the vesicle lipid bilayer most likely changed the intravesicular potential, thereby slowing the flux of ions through the fusion pore, hence reducing the rate of catecholamine secretion. These results suggest that catecholamine efflux is couple with the intravesicular membrane potential.

scholarly journals Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

1997 ◽  
Vol 136 (5) ◽  
pp. 995-1005 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sofya A. Brener ◽  
Dong C. Ok ◽  
Fredric S. Cohen
Keyword(s):  
Outer Membrane ◽  
Negative Curvature ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Positive Curvature ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

scholarly journals The Lipid-anchored Ectodomain of Influenza Virus Hemagglutinin (GPI-HA) Is Capable of Inducing Nonenlarging Fusion Pores

2000 ◽  
Vol 11 (4) ◽  
pp. 1143-1152 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Influenza Virus ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Initial Membrane ◽  
Optimal Fusion ◽  
Pass Through

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.

scholarly journals Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation

2002 ◽  
Vol 76 (9) ◽  
pp. 4603-4611 ◽  
Author(s):  
Tatsuya Sakai ◽  
Reiko Ohuchi ◽  
Masanobu Ohuchi
Keyword(s):  
Influenza Virus ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Reaction ◽  
Biological Significance ◽  
Site Directed Mutagenesis ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Calcein Leakage ◽  
Palmitic Acids

ABSTRACT Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Cathodoluminescence of Catalyst Crystallites

1982 ◽  
Vol 40 ◽  
pp. 664-665
Author(s):  
E.D. Boyes ◽  
P.L. Gai ◽  
D.B. Darby ◽  
C. Warwick
Keyword(s):  
Defect Density ◽  
Chemical Properties ◽  
Operating Conditions ◽  
Semiconductor Materials ◽  
Environmental Cell ◽  
High Resolution Structure ◽  
Commercially Important ◽  
Crystallographic Defects ◽  
Resolution Structure

The extended crystallographic defects introduced into some oxide catalysts under operating conditions may be a consequence and accommodation of the changes produced by the catalytic activity, rather than always being the origin of the reactivity. Operation without such defects has been established for the commercially important tellurium molybdate system. in addition it is clear that the point defect density and the electronic structure can both have a significant influence on the chemical properties and hence on the effectiveness (activity and selectivity) of the material as a catalyst. SEM/probe techniques more commonly applied to semiconductor materials, have been investigated to supplement the information obtained from in-situ environmental cell HVEM, ultra-high resolution structure imaging and more conventional AEM and EPMA chemical microanalysis.

Direct visualization of phase-separated domains in lipid membranes

1978 ◽  
Vol 36 (2) ◽  
pp. 290-291
Author(s):  
S. W. Hui ◽  
T. P. Stewart
Keyword(s):  
Lipid Membranes ◽  
Structural Information ◽  
Freeze Fracture ◽  
Biological Molecules ◽  
Vacuum Drying ◽  
Direct Visualization ◽  
X Ray Diffraction ◽  
Electron Microscopic ◽  
X Ray ◽  
Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

EMCAT: An automatic image-processing system for electron-microscopic tomography

1994 ◽  
Vol 52 ◽  
pp. 418-419
Author(s):  
Weiping Liu ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Image Processing ◽  
Structural Information ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Processing System ◽  
Electron Microscopic ◽  
Data Set ◽  
Ordered Structures ◽  
Automatic Image Processing ◽  
Em Tomography

Any real world object is three-dimensional. The principle of tomography, which reconstructs the 3-D structure of an object from its 2-D projections of different view angles has found application in many disciplines. Electron Microscopic (EM) tomography on non-ordered structures (e.g., subcellular structures in biology and non-crystalline structures in material science) has been exercised sporadically in the last twenty years or so. As vital as is the 3-D structural information and with no existing alternative 3-D imaging technique to compete in its high resolution range, the technique to date remains the kingdom of a brave few. Its tedious tasks have been preventing it from being a routine tool. One keyword in promoting its popularity is automation: The data collection has been automated in our lab, which can routinely yield a data set of over 100 projections in the matter of a few hours. Now the image processing part is also automated. Such automations finish the job easier, faster and better.

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