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- EMDB-18574: A segment of clathrin-coated endosome, isolated from mouse brain ... -

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Basic information

Entry
Database: EMDB / ID: EMD-18574
TitleA segment of clathrin-coated endosome, isolated from mouse brain tissue
Map data
Sample
  • Organelle or cellular component: Endosome
KeywordsCoated vesicles formation / ENDOCYTOSIS
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 151.8 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_18574.png

Downloads & links

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Map

FileDownload / File: emd_18574.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
13.44 Å/pix.
x 100 pix.
= 1344. Å		13.44 Å/pix.
x 100 pix.
= 1344. Å		13.44 Å/pix.
x 100 pix.
= 1344. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 13.44 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.487
Minimum - Maximum-2.3441336 - 3.754835
Average (Standard dev.)0.008787947 (±0.24583133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 1344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18574_msk_1.map
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Half map: #2

Fileemd_18574_half_map_1.map
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Half map: #1

Fileemd_18574_half_map_2.map
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Sample components

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Entire : Endosome

EntireName: Endosome
Components
  • Organelle or cellular component: Endosome

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Supramolecule #1: Endosome

SupramoleculeName: Endosome / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Endosome obtained from mouse brain synaptic vesicle isolation procedure
Source (natural)Organism: Mus musculus (house mouse) / Organ: Brain (hippocampus, cortex, and cerebellum) / Organelle: Endosome

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 98 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 6.2 e/Å2
Details: For several data collection, different total exposures were used: 128,270,229,271 e per A2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.5 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 53000
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 was acquired in the super-resolution mode (superresolution pixel size - 0.84 A). Data processing up to particle picking was performed using TomoBEAR (10.1101/2023.01.10.523437).
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 151.8 Å / Resolution method: FSC 0.5 CUT-OFF
Details: Due to the low amount of particles, even/odd maps were generated and the FSC was calculated without the refinement of each half-map against the reference with FSC 0.5 cut-off.
Number subtomograms used: 27
ExtractionNumber tomograms: 5 / Number images used: 1073 / Software - Name: Dynamo (ver. 1.1.532)
Final angle assignmentType: OTHER / Details: Cross-correlation
FSC plot (resolution estimation)

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