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- EMDB-18583: A segment of a clathrin-coated vesicle obtained from a primary hi... -

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Basic information

Entry
Database: EMDB / ID: EMD-18583
TitleA segment of a clathrin-coated vesicle obtained from a primary hippocampal neurons, cultured on grids
Map data
Sample
  • Organelle or cellular component: Primary hippocampal neuron culture
KeywordsENDOCYTOSIS
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 75.6 Å
AuthorsKravcenko U / Ruwolt M / Kroll J / Yushkevich A / Ruta J / Lotfy R / Rosenmund C / Liu F / Kudryashev M
Funding support Germany, 5 items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST 335/588-1 FUGG Titan Krios 300 keV Kryo-Transmissions-Elektronenmikroskop Germany
German Research Foundation (DFG)KU3221/3-1 Germany
German Research Foundation (DFG)458275811 Germany
German Research Foundation (DFG)399894546 Germany
German Research Foundation (DFG)436260754 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Molecular architecture of synaptic vesicles.
Authors: Uljana Kravčenko / Max Ruwolt / Jana Kroll / Artsemi Yushkevich / Martina Zenkner / Julia Ruta / Rowaa Lotfy / Erich E Wanker / Christian Rosenmund / Fan Liu / Mikhail Kudryashev /
Abstract: Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, ...Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs can be formed in a clathrin-dependent manner. This process maintains complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV (1). However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here, we used cryoelectron tomography to visualize molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram averaging, and showed them forming a complex with the membrane-embedded protein synaptophysin (Syp). Our bioluminescence assay revealed pairwise interactions between vesicle-associated membrane protein 2 and Syp and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle size. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with an icosahedral symmetry. We observed V-ATPases under clathrin cages in several isolated clathrin-coated vesicles (CCVs). Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their and CCVs preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs' diversity and their molecular architecture.
History
DepositionOct 3, 2023-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18583.png

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Map

FileDownload / File: emd_18583.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
24.56 Å/pix.
x 60 pix.
= 1473.6 Å		24.56 Å/pix.
x 60 pix.
= 1473.6 Å		24.56 Å/pix.
x 60 pix.
= 1473.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 24.56 Å
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Contour LevelBy AUTHOR: 2.05
Minimum - Maximum-7.8533916 - 15.230225000000001
Average (Standard dev.)0.009606695 (±0.68450004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 1473.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18583_msk_1.map
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Half map: #2

Fileemd_18583_half_map_1.map
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Half map: #1

Fileemd_18583_half_map_2.map
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Sample components

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Entire : Primary hippocampal neuron culture

EntireName: Primary hippocampal neuron culture
Components
  • Organelle or cellular component: Primary hippocampal neuron culture

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Supramolecule #1: Primary hippocampal neuron culture

SupramoleculeName: Primary hippocampal neuron culture / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Primary hippocampal neuron (17 DIV) cultured on grids
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R3.5/1 / Material: GOLD
VitrificationCryogen name: ETHANE
DetailsPrimary hippocampal neurons cultured on grids

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe data acquisition was performed in superresolution mode (superresolution pixel size = 3.07 A).
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 75.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 1564
ExtractionNumber tomograms: 34 / Number images used: 15789 / Software - Name: Dynamo (ver. 1.1.532)
Final angle assignmentType: OTHER / Software - Name: Dynamo (ver. 1.1.532) / Details: Cross-correlation
FSC plot (resolution estimation)

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