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- EMDB-18746: wild type neuronal synapse -

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Basic information

Entry
Database: EMDB / ID: EMD-18746
Titlewild type neuronal synapse
Map datawild type neuronal synapse. non filtered map.
Sample
  • Cell: Synapse between hippocampal neurons isolated from SNAP-25 KO and transduced with SNAP-25 wild type.
Keywordssynapse / neurotransmission / EXOCYTOSIS
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsRadecke J / Seeger R / Zuber B / Sorensen JB
Funding support Switzerland, Denmark, 5 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179520 Switzerland
Novo Nordisk FoundationNNF17OC0028516 Denmark
The Carlsberg FoundationCF17-0875 Denmark
Independent Research Fund Denmark - Technology and Production Sciences8020-00228A Denmark
LundbeckfondenR277-2018-802 Denmark
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_18746.png

Downloads & links

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Map

FileDownload / File: emd_18746.map.gz / Format: CCP4 / Size: 608 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotationwild type neuronal synapse. non filtered map.
Voxel sizeX=Y=Z: 14.69 Å
Density
Minimum - Maximum-352.0 - 291.0
Average (Standard dev.)3.7565894 (±18.416412000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-152
Dimensions10241024304
Spacing10241024304
CellA: 15042.56 Å / B: 15042.56 Å / C: 4465.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: wild type neuronal synapse. Non-linear anistropic diffusion filtered...

Fileemd_18746_additional_1.map
Annotationwild type neuronal synapse. Non-linear anistropic diffusion filtered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Synapse between hippocampal neurons isolated from SNAP-25 KO and ...

EntireName: Synapse between hippocampal neurons isolated from SNAP-25 KO and transduced with SNAP-25 wild type.
Components
  • Cell: Synapse between hippocampal neurons isolated from SNAP-25 KO and transduced with SNAP-25 wild type.

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Supramolecule #1: Synapse between hippocampal neurons isolated from SNAP-25 KO and ...

SupramoleculeName: Synapse between hippocampal neurons isolated from SNAP-25 KO and transduced with SNAP-25 wild type.
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57/Bl6 / Organ: Brain / Tissue: Hippocampus

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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
Details: NB medium (Neurobasal with 2% B-27, 1 M HEPES, 0.26% lutamax, 14.3 mM beta-mercaptoethanol, 10000 IU penicillin, 10 mg streptomycin)
GridModel: Quantifoil R2/2 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: After 12 to 14 days of incubation grids with mouse neurons were plunge frozen with a Vitrobot (Thermofisher Scientific, Mark IV) with a blot time of 3 s and a blot force of -10. Wait time ...Details: After 12 to 14 days of incubation grids with mouse neurons were plunge frozen with a Vitrobot (Thermofisher Scientific, Mark IV) with a blot time of 3 s and a blot force of -10. Wait time and drain time were not used. Humidity was set to 100% at 4 degrees C. 4 ul undiluted 10 nm BSA gold tracer (Aurion) was added directly onto the grid prior to plunge freezing..
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: AURION Immuno Gold Reagents & Accessories / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 10.0 µm / Nominal defocus min: 6.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 0.76 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: IMOD / Number images used: 131

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