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- EMDB-23077: Hexameric protein arrangement under docked synaptic vesicles in p... -

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Basic information

Entry
Database: EMDB / ID: EMD-23077
TitleHexameric protein arrangement under docked synaptic vesicles in primary hippocampal neurons
Map dataCryo-ET/ Subtomogram averaging of protein organization under docked synaptic vesicles from primary hippocampal neurons
Sample
  • Organelle or cellular component: Docked synaptic vesicles in primary hippocampal neurons
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 44.0 Å
AuthorsRadhakrishnan A / Li X / Liu J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK027044 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Symmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals.
Authors: Abhijith Radhakrishnan / Xia Li / Kirill Grushin / Shyam S Krishnakumar / Jun Liu / James E Rothman /
Abstract: Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV ...Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca influx. This Ca-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca influx.
History
DepositionDec 8, 2020-
Header (metadata) releaseFeb 3, 2021-
Map releaseFeb 3, 2021-
UpdateFeb 3, 2021-
Current statusFeb 3, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23077.png

Downloads & links

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Map

FileDownload / File: emd_23077.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-ET/ Subtomogram averaging of protein organization under docked synaptic vesicles from primary hippocampal neurons
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.8 Å/pix.
x 72 pix.
= 777.6 Å		10.8 Å/pix.
x 72 pix.
= 777.6 Å		10.8 Å/pix.
x 72 pix.
= 777.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 10.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-8.057052 - 6.1466413
Average (Standard dev.)-5.2878356e-08 (±1.0000737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 777.60004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.810.810.8
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z777.600777.600777.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-8.0576.147-0.000

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Supplemental data

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Sample components

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Entire : Docked synaptic vesicles in primary hippocampal neurons

EntireName: Docked synaptic vesicles in primary hippocampal neurons
Components
  • Organelle or cellular component: Docked synaptic vesicles in primary hippocampal neurons

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Supramolecule #1: Docked synaptic vesicles in primary hippocampal neurons

SupramoleculeName: Docked synaptic vesicles in primary hippocampal neurons
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Primary hippocampal neurons were cultured on EM grids
Source (natural)Organism: Mus musculus (house mouse) / Organ: Brain / Tissue: Hippocampus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 15 mA current
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Detailsprimary hippocampal neurons were cultured directly on EM grids

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.47 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 44.0 Å / Resolution method: FSC 0.5 CUT-OFF / Details: Subtomogram averaging / Number subtomograms used: 5726
ExtractionNumber tomograms: 296 / Number images used: 7527
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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