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- EMDB-2531: Tomographic subvolume average of EFF-1 fusogen on extracellular v... -

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Basic information

Entry
Database: EMDB / ID: EMD-2531
TitleTomographic subvolume average of EFF-1 fusogen on extracellular vesicles
Map dataSegmented membrane structure from EFF-1 coated vesicles
Sample
  • Sample: Segmented membrane from epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
  • Organelle or cellular component: Cell-derived vesicle membrane
Keywordscell-cell fusion / extracellular fusion / membrane fusion / fusogen / pre-fusion state
Biological speciesMesocricetus auratus (golden hamster)
Methodsubtomogram averaging / cryo EM
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: 3.46
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.46
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2531.png

Downloads & links

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Map

FileDownload / File: emd_2531.map.gz / Format: CCP4 / Size: 1.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSegmented membrane structure from EFF-1 coated vesicles
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 94 pix.
= 357.2 Å		3.8 Å/pix.
x 32 pix.
= 121.6 Å		3.8 Å/pix.
x 94 pix.
= 357.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 3.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.46 / Movie #1: 3.46
Minimum - Maximum0.0 - 17.90704727
Average (Standard dev.)1.21594417 (±2.61928034)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-47-47-47
Dimensions329494
Spacing329494
CellA: 357.19998 Å / B: 121.6 Å / C: 357.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z943294
origin x/y/z0.0000.0000.000
length x/y/z357.200121.600357.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-47-47-47
NC/NR/NS943294
D min/max/mean0.00017.9071.216

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Supplemental data

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Sample components

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Entire : Segmented membrane from epithelial fusion failure 1 (EFF-1) Isofo...

EntireName: Segmented membrane from epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
Components
  • Sample: Segmented membrane from epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
  • Organelle or cellular component: Cell-derived vesicle membrane

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Supramolecule #1000: Segmented membrane from epithelial fusion failure 1 (EFF-1) Isofo...

SupramoleculeName: Segmented membrane from epithelial fusion failure 1 (EFF-1) Isoform A on extracellular vesicles
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Cell-derived vesicle membrane

SupramoleculeName: Cell-derived vesicle membrane / type: organelle_or_cellular_component / ID: 1
Details: Membrane structure of EFF-1 coated extracellular vesicles
Recombinant expression: No
Source (natural)Organism: Mesocricetus auratus (golden hamster) / Strain: clone 13 / synonym: Golden Hamster / Tissue: Kidney / Cell: Fibroblast BHK 21 / Location in cell: Plasma membrane

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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
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
Specialist opticsEnergy filter - Name: Gatan Quantum 964 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 78950 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 95000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN HELIUM / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsSubtomograms were selected using a semi-automated picking algorithm
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Software - Name: PEET / Number subtomograms used: 1973
Final 3D classificationNumber classes: 1

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