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- EMDB-72056: Cryo ET of Native Hippocampal Glutamatergic Synapses without AuNP... -

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Basic information

Entry
Database: EMDB / ID: EMD-72056
TitleCryo ET of Native Hippocampal Glutamatergic Synapses without AuNP label
Map data
Sample
  • Tissue: Glutamatergic synapse from mouse hippocampus tissue
KeywordsGlutamatergic synapse / NEUROPEPTIDE
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsKim H / Spangler CJ / Matsui A / Elferich J / Gouaux E
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Dimeric gold nanoparticles enable multiplexed labeling in cryoelectron tomography.
Authors: Hoyoung Kim / Cathy J Spangler / Aya Matsui / Johannes Elferich / Junhoe Kim / Alex Roseborough / May Nyman / Tanja M Lahtinen / Eric Gouaux /
Abstract: Cryoelectron tomography (cryo-ET) enables three-dimensional visualization of molecular structures within tissue and intact cells, providing a powerful tool for studying the spatial organization of ...Cryoelectron tomography (cryo-ET) enables three-dimensional visualization of molecular structures within tissue and intact cells, providing a powerful tool for studying the spatial organization of biological components at nanometer resolution. Realizing this potential, particularly for submegadalton complexes, is facilitated by fiducial-based labeling. Gold nanoparticles (AuNPs) have emerged as powerful electron-dense labels for cryo-ET, but multiplexing using only conventional monomeric AuNPs is challenging, limiting their application in multitarget studies. Here, we describe functionalized dimeric AuNPs with a precisely defined size range applied to bioimaging, enabling multiplexed labeling by allowing reliable discrimination between monomeric and dimeric AuNPs, thereby supporting identification of distinct molecular targets within the same cryotomogram. Each dimer consists of two covalently linked gold particles of approximately 2.6 nm diameter separated by a defined spacing of about 1.4 nm, with high structural homogeneity validated by small-angle X-ray scattering (SAXS) and electron microscopy. The anti-GluN1 Fab, which targets the -methyl-D-aspartate receptor (NMDAR), was site specifically conjugated to a dimeric AuNP. A deep learning classifier enabled reliable discrimination between monomeric and dimeric AuNPs in tomograms. We confirmed that the dimeric AuNP-Fab conjugates bind robustly to recombinant GluN1/GluN2A receptors, validating their use for structural labeling. In situ cryo-ET of brain tissue further confirms that the dimeric labels reach NMDARs within the glutamatergic synaptic cleft. Combined with monomeric AuNPs, this dimeric AuNP platform establishes a generalizable approach for distinguishable labels optimized for cryo-ET. The compact size and structural uniformity of monomeric and dimeric AuNPs make them ideally suited for nanoscale molecular mapping in crowded cellular environments.
History
DepositionAug 8, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72056.png

Downloads & links

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Map

FileDownload / File: emd_72056.map.gz / Format: CCP4 / Size: 865.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10 Å/pix.
x 375 pix.
= 3750. Å		10 Å/pix.
x 778 pix.
= 7780. Å		10 Å/pix.
x 778 pix.
= 7780. 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: 10 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1.6436534 - 1.0504893
Average (Standard dev.)0.03935865 (±0.04175753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions778778375
Spacing778778375
CellA: 7780.0 Å / B: 7780.0 Å / C: 3750.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Glutamatergic synapse from mouse hippocampus tissue

EntireName: Glutamatergic synapse from mouse hippocampus tissue
Components
  • Tissue: Glutamatergic synapse from mouse hippocampus tissue

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Supramolecule #1: Glutamatergic synapse from mouse hippocampus tissue

SupramoleculeName: Glutamatergic synapse from mouse hippocampus tissue / type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 / Organ: Brain / Tissue: Hippocampus

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
GridModel: EMS Lacey Carbon / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: NITROGEN
High pressure freezingInstrument: OTHER
Details: The value given for _em_high_pressure_freezing.instrument is Leica ICE. This is not in a list of allowed values {'LEICA EM PACT2', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'EMS-002 RAPID ...Details: The value given for _em_high_pressure_freezing.instrument is Leica ICE. This is not in a list of allowed values {'LEICA EM PACT2', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100'} so OTHER is written into the XML file.
Cryo protectant20 % dextran /5 % sucrose
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: 120 / Focused ion beam - Temperature: 83 K / Focused ion beam - Initial thickness: 40000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
Fiducial markerManufacturer: Gikd nanoparticle / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 33
CTF correctionSoftware - Name: MotionCorr2 / Type: NONE

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