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- EMDB-8344: Influenza virus membrane fusion with liposomes -

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Basic information

Entry
Database: EMDB / ID: EMD-8344
TitleInfluenza virus membrane fusion with liposomes
Map dataInfluenza virus membrane fusion with liposomes
Sample
  • Virus: Influenza A virus
Biological speciesInfluenza A virus
Methodelectron tomography / cryo EM
AuthorsGui L / Lee K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM099989 United States
CitationJournal: J Virol / Year: 2016
Title: Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion.
Authors: Long Gui / Jamie L Ebner / Alexander Mileant / James A Williams / Kelly K Lee /
Abstract: Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of ...Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved.
IMPORTANCE: Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is ...IMPORTANCE: Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.
History
DepositionAug 22, 2016-
Header (metadata) releaseFeb 8, 2017-
Map releaseFeb 8, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8344.png

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Map

FileDownload / File: emd_8344.map.gz / Format: CCP4 / Size: 905.5 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationInfluenza virus membrane fusion with liposomes
Voxel sizeX=Y=Z: 3.19 Å
Density
Minimum - Maximum-127 - 125
Average (Standard dev.)-6.2495627 (±100.52)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions16681725330
Spacing17251668330
CellA: 5502.75 Å / B: 5320.92 Å / C: 1052.7001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z3.193.193.19
M x/y/z17251668330
origin x/y/z0.0000.0000.000
length x/y/z5502.7505320.9201052.700
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS17251668330
D min/max/mean-127.000125.000-6.250

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Supplemental data

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Sample components

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Entire : Influenza A virus

EntireName: Influenza A virus
Components
  • Virus: Influenza A virus

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Supramolecule #1: Influenza A virus

SupramoleculeName: Influenza A virus / type: virus / ID: 1 / Parent: 0
Details: X31 (H3N2) influenza A virus grown in embryonated chicken eggs was purchased from Charles River Lab.
NCBI-ID: 11320 / Sci species name: Influenza A virus / Sci species strain: X31 (H3N2) / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 5.5
Component:
ConcentrationFormulaName
150.0 mg/mLNaClSodium chloridesodium chloride
10.0 mg/mLC8H18N2O4SHEPES
50.0 mg/mLNa3C6H5O7sodium citrate
GridModel: C-flat / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 25 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters of solution was added to glow-discharged holey carbon-coated grids (C-flat, 200 mesh, Electron Microscopy Sciences) and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV..
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Electron Microscopy Sciences / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 11500
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-11 / Average exposure time: 1.7 sec. / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.7) / Number images used: 60

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