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- EMDB-18744: Stimulated rat synaptosome. -

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Basic information

Entry
Database: EMDB / ID: EMD-18744
TitleStimulated rat synaptosome.
Map datastimulated rat synaptosome
Sample
  • Cell: Stimulated rat synaptosome
Keywordssynapse / neurotransmission / EXOCYTOSIS
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsRadecke J / Seeger R / Zuber B
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179520 Switzerland
CitationJournal: EMBO Rep / Year: 2023
Title: Morphofunctional changes at the active zone during synaptic vesicle exocytosis.
Authors: Julika Radecke / Raphaela Seeger / Anna Kádková / Ulrike Laugks / Amin Khosrozadeh / Kenneth N Goldie / Vladan Lučić / Jakob B Sørensen / Benoît Zuber /
Abstract: Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate ...Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.
History
DepositionOct 25, 2023-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateNov 8, 2023-
Current statusNov 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18744.png

Downloads & links

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Map

FileDownload / File: emd_18744.map.gz / Format: CCP4 / Size: 126.8 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotationstimulated rat synaptosome
Voxel sizeX=Y=Z: 22.4 Å
Density
Minimum - Maximum-11278.0 - 5441.0
Average (Standard dev.)209.149020000000007 (±220.108630000000005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-153-15188
Dimensions656576176
Spacing576656176
CellA: 12902.399 Å / B: 14694.399 Å / C: 3942.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Stimulated rat synaptosome

EntireName: Stimulated rat synaptosome
Components
  • Cell: Stimulated rat synaptosome

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Supramolecule #1: Stimulated rat synaptosome

SupramoleculeName: Stimulated rat synaptosome / type: cell / ID: 1 / Parent: 0
Details: sprayed with KCl milliseconds before freezing in order to stimulate exocytosis.
Source (natural)Organism: Rattus norvegicus (Norway rat) / Organ: Brain / Tissue: Cortex

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
140.0 mMNaClSodium chloridesodium chloride
52.0 mMKClpotassium chloride
5.0 mMNaHCO3sodium bicarbonate
1.2 mMNa2HPO4disodium hydrogen phosphate
1.0 mMMgCl2magnesium chloride
10.0 mMC6H12O6glucose
20.0 mMC8H18N2O4SHEPES
1.0 mMCaCl2calcium chloride
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: AURION Immuno Gold Reagents & Accessories / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 12.0 µm / Nominal defocus min: 8.0 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 1.96 e/Å2

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 101

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