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- EMDB-44174: Cryo-electron tomographic investigation of native hippocampal glu... -

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Basic information

Entry
Database: EMDB / ID: EMD-44174
TitleCryo-electron tomographic investigation of native hippocampal glutamatergic synapses - Tomogram 1
Map dataTomogram of glutamatergic synapse
Sample
  • Tissue: Glutamatergic synapse from mouse hippocampus tissue
KeywordsSynapse AMPA Neurotransmission / MEMBRANE PROTEIN
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsMatsui A / Spangler CJ / Elferich J / Gouaux E
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2024
Title: Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses.
Authors: Aya Matsui / Cathy Spangler / Johannes Elferich / Momoko Shiozaki / Nikki Jean / Xiaowei Zhao / Maozhen Qin / Haining Zhong / Zhiheng Yu / Eric Gouaux /
Abstract: Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary ...Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here, we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for the identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft, and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.
History
DepositionMar 20, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44174.png

Downloads & links

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Map

FileDownload / File: emd_44174.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of glutamatergic synapse
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10 Å/pix.
x 250 pix.
= 2500. Å		10 Å/pix.
x 1250 pix.
= 12500. Å		10 Å/pix.
x 1000 pix.
= 10000. 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-4.3566175 - 2.401464
Average (Standard dev.)0.024149587 (±0.1699363)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-8137-50
Dimensions12501000250
Spacing10001250250
CellA: 10000.0 Å / B: 12500.0 Å / C: 2500.0 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: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: at 15 mA
VitrificationCryogen name: NITROGEN
DetailsDissected hippocampus tissue from vGlut1-mScarlet/PSD95-EGFP mouse line
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 {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM PACT', 'LEICA EM HPM100', 'LEICA ...Details: The value given for _em_high_pressure_freezing.instrument is Leica Ice. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM PACT', 'LEICA EM HPM100', 'LEICA EM PACT2', 'BAL-TEC HPM 010', 'OTHER'} 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 4.55 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: 2.5 µm / Nominal defocus min: 2.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: RELION (ver. 4.0.1) / Number images used: 33

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