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- EMDB-45161: Cryo-Electron Tomography of Cultured Neuronal Synapses -

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Basic information

Entry
Database: EMDB / ID: EMD-45161
TitleCryo-Electron Tomography of Cultured Neuronal Synapses
Map dataCryoCARE denoised tomogram of a cultured neuronal synapse.
Sample
  • Cell: Tomograms of Cultured Neuronal Synapses
KeywordsSynapse / Neuron / Vesicles / EXOCYTOSIS
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsHeld RG / Brunger AT / Liang J
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH063105 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography.
Authors: Richard G Held / Jiahao Liang / Axel T Brunger /
Abstract: The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in ...The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.
History
DepositionMay 31, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45161.png

Downloads & links

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Map

FileDownload / File: emd_45161.map.gz / Format: CCP4 / Size: 120.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.6 Å/pix.
x 86 pix.
= 1169.6 Å		13.6 Å/pix.
x 720 pix.
= 9792. Å		13.6 Å/pix.
x 512 pix.
= 6963.2 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: 13.6 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-133.228360000000009 - 127.245099999999994
Average (Standard dev.)36.529240000000001 (±5.163511)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125104-70
Dimensions72051286
Spacing51272086
CellA: 6963.2 Å / B: 9792.0 Å / C: 1169.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_1.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_10.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_11.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_12.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_2.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_3.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_4.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_5.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_6.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_7.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_8.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: CryoCARE denoised tomogram of a cultured neuronal synapse.

Fileemd_45161_additional_9.map
AnnotationCryoCARE denoised tomogram of a cultured neuronal synapse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tomograms of Cultured Neuronal Synapses

EntireName: Tomograms of Cultured Neuronal Synapses
Components
  • Cell: Tomograms of Cultured Neuronal Synapses

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Supramolecule #1: Tomograms of Cultured Neuronal Synapses

SupramoleculeName: Tomograms of Cultured Neuronal Synapses / type: cell / ID: 1 / Parent: 0
Details: Tomograms of cryo-FIB lamellae of from cultured neurons.
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6J / 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
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Detailstomograms from FIB-milled lamellae of cultured mouse hippocampal neurons, targeting neuronal synapses.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 600 / Focused ion beam - Temperature: 83 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 175
Focused ion beam - Details: The milling sequence was the following (all at 30 keV): rough milling: 0.3 nA, 3 um pattern separation in Y; medium milling: 0.1 nA, 1 um pattern separation; fine milling: ...Focused ion beam - Details: The milling sequence was the following (all at 30 keV): rough milling: 0.3 nA, 3 um pattern separation in Y; medium milling: 0.1 nA, 1 um pattern separation; fine milling: 50 pA, 500 nm pattern separation; polishing: 30 pA, 175 nm pattern separation.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.
Fiducial markerDiameter: 2.5 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 3.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 9.0 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

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD
Details: IMOD was used for tilt-series alignment and weighted back-projection, followed by denoising using CryoCARE.
Number images used: 41

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