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- EMDB-6210: Structure of 20S supercomplex with V7-SNARE determined by single ... -

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Entry
Database: EMDB / ID: 6210
TitleStructure of 20S supercomplex with V7-SNARE determined by single particle cryoelectron microscopy
Map dataMap of 20S supercomplex with V7-SNARE. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
Sample20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
  • N-ethylmaleimide sensitive factor
  • alpha Soluble NSF Attachment Protein
  • Syntaxin-1A
  • Vesicle-associated membrane protein 7
  • Synaptosomal-associated protein 25SNAP25
Keywordsvesicle trafficking
Function / homologySynaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / SNARE domain / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) / Target SNARE coiled-coil homology domain ...Synaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / SNARE domain / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) / Target SNARE coiled-coil homology domain / NSF attachment protein / Vesicle-fusing ATPase / SNAP-25 / Syntaxin 1A / CDC48, N-terminal subdomain / P-loop containing nucleoside triphosphate hydrolase / Synaptobrevin / Tetratricopeptide-like helical domain superfamily / Longin-like domain superfamily / SNARE / Longin domain / Aspartate decarboxylase-like domain superfamily / Syntaxin/epimorphin, conserved site / Syntaxin, N-terminal domain / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / Regulated-SNARE-like domain / Synaptobrevin signature. / AAA-protein family signature. / Syntaxin / epimorphin family signature. / Interleukin-12 signaling / GABA synthesis, release, reuptake and degradation / Clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / Retrograde transport at the Trans-Golgi-Network / Intra-Golgi traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / LGI-ADAM interactions / Other interleukin signaling / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / Clathrin derived vesicle budding / Acetylcholine Neurotransmitter Release Cycle / Dopamine Neurotransmitter Release Cycle / Glutamate Neurotransmitter Release Cycle / COPII-mediated vesicle transport / Norepinephrine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / v-SNARE coiled-coil homology domain profile. / Longin domain profile. / t-SNARE coiled-coil homology domain profile. / AAA+ ATPase domain / regulation of protein targeting to vacuolar membrane / natural killer cell degranulation / positive regulation of histamine secretion by mast cell / soluble NSF attachment protein activity / triglyceride transport / eosinophil degranulation / SNARE complex disassembly / myosin head/neck binding / regulation of synaptic vesicle priming / anchored component of presynaptic membrane / vesicle fusion with Golgi apparatus / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / neurotransmitter transport / positive regulation of norepinephrine secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / presynaptic active zone membrane / synaptic vesicle fusion to presynaptic active zone membrane / positive regulation of catecholamine secretion / synaptic vesicle docking / SNARE complex assembly / synaptic vesicle priming / vesicle transport along microtubule / intracellular organelle / positive regulation of neurotransmitter secretion / apical protein localization / protein-containing complex disassembly / positive regulation of calcium ion-dependent exocytosis / calcium ion-regulated exocytosis of neurotransmitter / calcium ion regulated exocytosis / regulation of synapse assembly / vesicle docking / vesicle-fusing ATPase / Golgi to plasma membrane protein transport / protein localization to membrane / chloride channel inhibitor activity / vesicle fusion / response to gravity / integral component of synaptic vesicle membrane / positive regulation of hormone secretion / SNAP receptor activity / sleep / positive regulation of ATPase activity / platelet alpha granule / synaptic vesicle exocytosis
Function and homology information
SourceCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / 8 Å resolution
AuthorsZhao M / Wu S / Zhou Q / Vivona S / Cipriano DJ / Cheng Y / Brunger AT
CitationJournal: Nature / Year: 2015
Title: Mechanistic insights into the recycling machine of the SNARE complex.
Authors: Minglei Zhao / Shenping Wu / Qiangjun Zhou / Sandro Vivona / Daniel J Cipriano / Yifan Cheng / Axel T Brunger
DateDeposition: Dec 5, 2014 / Header (metadata) release: Jan 28, 2015 / Map release: Jan 28, 2015 / Last update: Feb 11, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6210.map.gz (map file in CCP4 format, 8193 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
2.43 Å/pix.
= 311.194 Å
128 pix
2.43 Å/pix.
= 311.194 Å
128 pix
2.43 Å/pix.
= 311.194 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4312 Å
Density
Contour Level:5 (by author), 5 (movie #1):
Minimum - Maximum-4.03881359 - 12.52900982
Average (Standard dev.)0E-8 (1.00000000)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin-64-64-64
Limit636363
Spacing128128128
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.4312031252.4312031252.431203125
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z311.194311.194311.194
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-4.03912.529-0.000

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Supplemental data

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Sample components

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Entire 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, ...

EntireName: 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
Number of components: 5
Oligomeric State: One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex

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Component #1: protein, N-ethylmaleimide sensitive factor

ProteinName: N-ethylmaleimide sensitive factor / a.k.a: NSF / Oligomeric Details: hexamer / Number of Copies: 6 / Recombinant expression: Yes
MassTheoretical: 83 kDa
SourceSpecies: Cricetulus griseus (Chinese hamster)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pROEX-1 / Strain: BL21-DE3-RIL
External referencesUniProt: Vesicle-fusing ATPase

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Component #2: protein, alpha Soluble NSF Attachment Protein

ProteinName: alpha Soluble NSF Attachment Protein / a.k.a: alpha-SNAP / Recombinant expression: Yes / Number of Copies: 4
MassTheoretical: 33 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pJ414 / Strain: BL21(DE3)
External referencesUniProt: Alpha-soluble NSF attachment protein

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Component #3: protein, Syntaxin-1A

ProteinName: Syntaxin-1A / a.k.a: Stx-1A / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 30 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pACYC-DUET-1 / Strain: BL21(DE3)
External referencesUniProt: Syntaxin-1A

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Component #4: protein, Vesicle-associated membrane protein 7

ProteinName: Vesicle-associated membrane protein 7 / a.k.a: VAMP-7 / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 22 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pACYC-DUET-1 / Strain: BL21(DE3)
External referencesUniProt: Vesicle-associated membrane protein 7

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Component #5: protein, Synaptosomal-associated protein 25

ProteinName: Synaptosomal-associated protein 25SNAP25 / a.k.a: SNAP-25SNAP25 / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 25 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET-DUET-1 / Strain: BL21(DE3)
External referencesUniProt: Synaptosomal-associated protein 25

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 15 mg/ml
Buffer solution: 50 mM Tris-Cl, 150 mM NaCl, 1 mM ATP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
pH: 8
Support filmHoley carbon on top of 400 mesh copper grid
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 90 K / Humidity: 100 % / Method: Blot for 3.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: May 12, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 44 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000 X (nominal) / Cs: 2.3 mm / Imaging mode: BRIGHT FIELD / Defocus: -1800 - -2800 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionDetails: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 32100
Details: 3D classification, refinement, and reconstruction were performed using RELION.
3D reconstructionSoftware: RELION / CTF correction: Each particle / Resolution: 8 Å / Resolution method: FSC 0.143, gold-standard

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