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- EMDB-2530: Tomographic subvolume average of EFF-1 fusogen on extracellular v... -

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Basic information

Entry
Database: EMDB / ID: EMD-2530
TitleTomographic subvolume average of EFF-1 fusogen on extracellular vesicles
Map dataTomographic subvolume average of EFF-1 fusion protein on extracellular vesicles
Sample
  • Sample: Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
  • Protein or peptide: Epithelial fusion failure 1, isoform a
Keywordscell-cell fusion / extracellular fusion / membrane fusion / fusogen / pre-fusion state
Function / homology
Function and homology information


nematode male tail mating organ morphogenesis / fusogenic activity / EFF-1 complex / chordate pharyngeal muscle development / post-embryonic body morphogenesis / nematode male tail tip morphogenesis / plasma membrane fusion / vulval development / cell-cell fusion / syncytium formation by plasma membrane fusion ...nematode male tail mating organ morphogenesis / fusogenic activity / EFF-1 complex / chordate pharyngeal muscle development / post-embryonic body morphogenesis / nematode male tail tip morphogenesis / plasma membrane fusion / vulval development / cell-cell fusion / syncytium formation by plasma membrane fusion / embryonic body morphogenesis / egg-laying behavior / cell-cell contact zone / locomotion / plasma membrane => GO:0005886 / morphogenesis of an epithelium / kinase activity / phosphorylation / extracellular region / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Cell-cell fusogen EFF/AFF / Cell-cell fusogen EFF/AFF, domain 3 / Type I membrane glycoproteins cell-cell fusogen
Similarity search - Domain/homology
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsubtomogram averaging / cryo EM / Resolution: 22.2 Å
AuthorsZeev-Ben-Mordehai T / Vasishtan D / Siebert CA / Grunewald K
CitationJournal: Nat Commun / Year: 2014
Title: The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane.
Authors: Tzviya Zeev-Ben-Mordehai / Daven Vasishtan / C Alistair Siebert / Kay Grünewald /
Abstract: Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, ...Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins.
History
DepositionDec 17, 2013-
Header (metadata) releaseJan 15, 2014-
Map releaseAug 27, 2014-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 18.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 18.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cyl
  • Surface level: 18.8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2530.png

Downloads & links

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Map

FileDownload / File: emd_2530.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomographic subvolume average of EFF-1 fusion protein on extracellular vesicles
Voxel sizeX=Y=Z: 3.8 Å
Density
Contour LevelBy AUTHOR: 18.800000000000001 / Movie #1: 18.8
Minimum - Maximum-38.063011170000003 - 34.33685303
Average (Standard dev.)-2.66537809 (±3.18243027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z342.000342.000342.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-38.06334.337-2.665

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Supplemental data

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Sample components

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Entire : Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular ve...

EntireName: Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
Components
  • Sample: Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
  • Protein or peptide: Epithelial fusion failure 1, isoform a

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Supramolecule #1000: Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular ve...

SupramoleculeName: Epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 1
Molecular weightTheoretical: 87 KDa

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Macromolecule #1: Epithelial fusion failure 1, isoform a

MacromoleculeName: Epithelial fusion failure 1, isoform a / type: protein_or_peptide / ID: 1 / Name.synonym: EFF-1a / Details: Proteins on extracellular vesicles / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / synonym: Golden Hamster / Cell: BHK 21 / Organelle: Extracellular vesicles / Location in cell: Extracellular vesicles
Molecular weightTheoretical: 87 KDa
Recombinant expressionOrganism: Mesocricetus auratus (golden hamster) / Recombinant cell: BHK 21 / Recombinant plasmid: pCAGGS
SequenceUniProtKB: EFF-1A
GO: plasma membrane fusion, syncytium formation by plasma membrane fusion, plasma membrane => GO:0005886, extracellular region, identical protein binding

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 25mM HEPES, 130mM NaCl
GridDetails: Holey carbon on top of 200 mesh gold grid.
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 78950 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 95000
Specialist opticsEnergy filter - Name: Gatan Quantum 964 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN HELIUM / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureMin: 80 K / Max: 100 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 115,000 times magnification
Legacy - Electron beam tilt params: +1 to -1 mradians
DateMar 3, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 30 µm / Number real images: 78 / Average electron dose: 60 e/Å2 / Details: 2 tomograms each created from 39 projection images / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final 3D classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.2 Å / Resolution method: OTHER / Software - Name: PEET / Number subtomograms used: 801
DetailsSubtomograms were selected using a semi-automated picking algorithm
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1ok8


Chain - Chain ID: A
SoftwareName: Chimera
DetailsFitting of a class II dengue virus E protein (monomer) to show similarity to class II fusion proteins
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4cyl:
Tomographic subvolume average of EFF-1 fusogen on extracellular vesicles

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