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- EMDB-0413: Vesicle-plasma interface under the docked vesicles in NGF-differe... -

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Basic information

Entry
Database: EMDB / ID: EMD-0413
TitleVesicle-plasma interface under the docked vesicles in NGF-differentiated PC12 cells without imposed symmetry
Map dataVesicle-plasma interface under the docked vesicles in NGF-differentiated PC12 cells without imposed symmetry
Sample
  • Organelle or cellular component: Docked synaptic vesicle
Biological speciesRattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM / Resolution: 50.0 Å
AuthorsLi X / Radhakrishnan A / Grushin K / Krishnakumar S / Liu J / Rothman J
CitationJournal: FEBS Lett / Year: 2019
Title: Symmetrical organization of proteins under docked synaptic vesicles.
Authors: Xia Li / Abhijith Radhakrishnan / Kirill Grushin / Ravikiran Kasula / Arunima Chaudhuri / Sujatha Gomathinayagam / Shyam S Krishnakumar / Jun Liu / James E Rothman /
Abstract: During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this ...During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.
History
DepositionDec 8, 2018-
Header (metadata) releaseJan 9, 2019-
Map releaseJan 9, 2019-
UpdateFeb 13, 2019-
Current statusFeb 13, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0413.png

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Map

FileDownload / File: emd_0413.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVesicle-plasma interface under the docked vesicles in NGF-differentiated PC12 cells without imposed symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.4 Å/pix.
x 120 pix.
= 648. Å		5.4 Å/pix.
x 120 pix.
= 648. Å		5.4 Å/pix.
x 120 pix.
= 648. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-4.1466355 - 5.9416695
Average (Standard dev.)-0.00021422854 (±0.9559543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 648.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.45.45.4
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z648.000648.000648.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-4.1475.942-0.000

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Supplemental data

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Sample components

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Entire : Docked synaptic vesicle

EntireName: Docked synaptic vesicle
Components
  • Organelle or cellular component: Docked synaptic vesicle

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Supramolecule #1: Docked synaptic vesicle

SupramoleculeName: Docked synaptic vesicle / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Synaptic vesicles in neurites developed from nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells
Source (natural)Organism: Rattus norvegicus (Norway rat) / Cell: PC12

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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
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / 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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 2434
ExtractionNumber tomograms: 574 / Number images used: 4758
Final angle assignmentType: OTHER

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