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

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Entry
Database: EMDB / ID: EMD-6210
TitleStructure of 20S supercomplex with V7-SNARE determined by single particle cryoelectron microscopy
Map data
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 / homology
Function and homology information


regulation of protein targeting to vacuolar membrane / natural killer cell degranulation / positive regulation of histamine secretion by mast cell / triglyceride transport / soluble NSF attachment protein activity / SNARE complex disassembly / vesicle fusion with Golgi apparatus / myosin head/neck binding / anchored component of presynaptic membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane ...regulation of protein targeting to vacuolar membrane / natural killer cell degranulation / positive regulation of histamine secretion by mast cell / triglyceride transport / soluble NSF attachment protein activity / SNARE complex disassembly / vesicle fusion with Golgi apparatus / myosin head/neck binding / anchored component of presynaptic membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / eosinophil degranulation / hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / neurotransmitter transport / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex / regulation of synaptic vesicle priming / positive regulation of norepinephrine secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptic vesicle fusion to presynaptic active zone membrane / presynaptic active zone membrane / positive regulation of catecholamine secretion / short-term synaptic potentiation / regulation of establishment of protein localization / vesicle transport along microtubule / synaptic vesicle docking / regulated exocytosis / protein-containing complex disassembly / intracellular organelle / calcium ion-regulated exocytosis of neurotransmitter / neurotransmitter receptor internalization / vesicle docking / secretion by cell / positive regulation of calcium ion-dependent exocytosis / apical protein localization / protein localization to membrane / membrane fusion / positive regulation of neurotransmitter secretion / integral component of synaptic vesicle membrane / vesicle fusion / Golgi to plasma membrane protein transport / vesicle-fusing ATPase / positive regulation of hormone secretion / SNARE complex / SNAP receptor activity / SNARE complex assembly / chloride channel inhibitor activity / response to gravity / calcium-ion regulated exocytosis / platelet alpha granule / synaptic vesicle priming / regulation of exocytosis / positive regulation of ATPase activity / positive regulation of synaptic plasticity / endosome to lysosome transport / positive regulation of receptor recycling / regulation of synapse assembly / ATP-dependent protein binding / regulation of neuron projection development / actomyosin / sleep / syntaxin-1 binding / azurophil granule membrane / vacuolar membrane / synaptic vesicle exocytosis / modulation of excitatory postsynaptic potential / positive regulation of dendrite morphogenesis / myosin binding / neurotransmitter secretion / positive regulation of exocytosis / insulin secretion / syntaxin binding / protein sumoylation / pseudopodium / voltage-gated potassium channel complex / exocytosis / ionotropic glutamate receptor binding / voltage-gated potassium channel activity / synaptic vesicle endocytosis / endomembrane system / associative learning / late endosome membrane / calcium channel inhibitor activity / long-term memory / positive regulation of insulin secretion / somatodendritic compartment / positive regulation of excitatory postsynaptic potential / phagocytic vesicle / SNARE binding / long-term synaptic potentiation / transport vesicle / axonal growth cone / synaptic vesicle membrane / photoreceptor inner segment / axonogenesis / acrosomal vesicle / synaptic transmission, glutamatergic / locomotory behavior / vesicle-mediated transport / presynapse
Longin domain / AAA+ ATPase domain / Longin-like domain superfamily / Syntaxin 1 / P-loop containing nucleoside triphosphate hydrolase / Tetratricopeptide-like helical domain superfamily / CDC48 domain 2-like superfamily / Target SNARE coiled-coil homology domain / Synaptosomal-associated protein 25 / SNAP-25 domain ...Longin domain / AAA+ ATPase domain / Longin-like domain superfamily / Syntaxin 1 / P-loop containing nucleoside triphosphate hydrolase / Tetratricopeptide-like helical domain superfamily / CDC48 domain 2-like superfamily / Target SNARE coiled-coil homology domain / Synaptosomal-associated protein 25 / SNAP-25 domain / Synaptobrevin / CDC48, N-terminal subdomain / NSF attachment protein / ATPase, AAA-type, core / Vesicle-fusing ATPase / AAA ATPase, AAA+ lid domain / v-SNARE, coiled-coil homology domain / Aspartate decarboxylase-like domain superfamily / ATPase, AAA-type, conserved site / Syntaxin/epimorphin, conserved site / CDC48, domain 2 / Syntaxin, N-terminal domain / SNARE
Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 7
Biological speciesCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 8 Å
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 /
Abstract: Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N- ...Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
History
Current status-Processing site: RCSB / Status: Released
DepositionDec 5, 2014-
Header (metadata) releaseJan 28, 2015-
Map releaseJan 28, 2015-
UpdateFeb 11, 2015-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • 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

emd_6210.png

Downloads & links

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Map

FileDownload / File: emd_6210.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-4.03881359 - 12.529009820000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 °

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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