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- EMDB-16085: Munc13-SNAP25 cryo-ET dataset, synapse tomo Munc13 DKO 102 (id: m... -

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Basic information

Entry
Database: EMDB / ID: EMD-16085
TitleMunc13-SNAP25 cryo-ET dataset, synapse tomo Munc13 DKO 102 (id: m13_dko_102)
Map dataSynapse Munc13 DKO 102 (id: m13_dko_102)
Sample
  • Organelle or cellular component: Synaptosome
KeywordsNeuronal synapse / Presynaptic terminal / Tethering / SNARE complex / CELL ADHESION
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsPapantoniou C / Lucic V
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)LU 1819/2-1 Germany
CitationJournal: Sci Adv / Year: 2023
Title: Munc13- and SNAP25-dependent molecular bridges play a key role in synaptic vesicle priming.
Authors: Christos Papantoniou / Ulrike Laugks / Julia Betzin / Cristina Capitanio / José Javier Ferrero / José Sánchez-Prieto / Susanne Schoch / Nils Brose / Wolfgang Baumeister / Benjamin H ...Authors: Christos Papantoniou / Ulrike Laugks / Julia Betzin / Cristina Capitanio / José Javier Ferrero / José Sánchez-Prieto / Susanne Schoch / Nils Brose / Wolfgang Baumeister / Benjamin H Cooper / Cordelia Imig / Vladan Lučić /
Abstract: Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies ...Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble -ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.
History
DepositionNov 2, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 12, 2023-
Current statusJul 12, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16085.png

Downloads & links

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Map

FileDownload / File: emd_16085.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSynapse Munc13 DKO 102 (id: m13_dko_102)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.56 Å/pix.
x 400 pix.
= 7024. Å		17.56 Å/pix.
x 928 pix.
= 16295.68 Å		17.56 Å/pix.
x 928 pix.
= 16295.68 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 17.56 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-15000.019000000000233 - 15000.019000000000233
Average (Standard dev.)680.202300000000037 (±563.933300000000031)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-46
Dimensions928928400
Spacing928928400
CellA: 16295.68 Å / B: 16295.68 Å / C: 7024.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Synaptosome

EntireName: Synaptosome
Components
  • Organelle or cellular component: Synaptosome

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Supramolecule #1: Synaptosome

SupramoleculeName: Synaptosome / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Central nervous system excitatiory synapse from a Munc13-1/2 double homozygous knockout mouse (Munc13 -/- -/-)
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57 B6/N / Organ: brain / Tissue: hippocampus / Organelle: excitatory synapse

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.4 / Component - Concentration: 300.0 mM / Component - Formula: HBM / Component - Name: Hepes-buffered medium
Details: 140 mM NaCl, 5 mM KCl, 5mM NaHCO3, 1.2 mM NaH2PO4-H2O, 1 mM MgCl26-H2O, 10 mM Glucose, 10 mM Hepes, 1.2 mM CaCl2
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Portable manual plunger made at Max Planck Institute of Biochemistry.
DetailsSynaptosomal fraction (P2) was obtained from DIV 28-30 hippocampal organotypic slices. These hippocampal organotypic slices were obtained from E18 mice.
Cryo protectantNone
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Slit width: 20 eV
SoftwareName: SerialEM
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.5 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

Final reconstructionAlgorithm: BACK PROJECTION / Number images used: 60

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