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Entry
Database: EMDB / ID: EMD-6206
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy, state I
Map dataMap of 20S supercomplex, state I. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
Sample
  • Sample: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
  • Protein or peptide: N-ethylmaleimide sensitive factor
  • Protein or peptide: alpha Soluble NSF Attachment Protein
  • Protein or peptide: Syntaxin-1A
  • Protein or peptide: Synaptobrevin-2
  • Protein or peptide: Synaptosomal-associated protein 25
Keywordsvesicle trafficking
Function / homology
Function and homology information


synaptic vesicle protein transport vesicle / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / trans-Golgi Network Vesicle Budding / regulation of delayed rectifier potassium channel activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / soluble NSF attachment protein activity / lamellar body ...synaptic vesicle protein transport vesicle / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / trans-Golgi Network Vesicle Budding / regulation of delayed rectifier potassium channel activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / soluble NSF attachment protein activity / lamellar body / BLOC-1 complex / short-term synaptic potentiation / Lysosome Vesicle Biogenesis / myosin head/neck binding / positive regulation of voltage-gated calcium channel activity / zymogen granule membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / calcium-dependent activation of synaptic vesicle fusion / presynaptic dense core vesicle exocytosis / extrinsic component of presynaptic membrane / calcium ion-regulated exocytosis of neurotransmitter / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / storage vacuole / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / protein-containing complex disassembly / positive regulation of catecholamine secretion / regulation of establishment of protein localization / positive regulation of norepinephrine secretion / Dopamine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / hormone secretion / regulation of synaptic vesicle priming / Golgi Associated Vesicle Biogenesis / : / regulated exocytosis / eosinophil degranulation / secretion by cell / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / positive regulation of glutamate secretion, neurotransmission / calcium-ion regulated exocytosis / SNARE complex disassembly / positive regulation of calcium ion-dependent exocytosis / positive regulation of hormone secretion / positive regulation of intracellular protein transport / positive regulation of neurotransmitter secretion / ribbon synapse / regulation of vesicle-mediated transport / : / positive regulation of vesicle fusion / Cargo recognition for clathrin-mediated endocytosis / chloride channel inhibitor activity / Clathrin-mediated endocytosis / regulation of exocytosis / protein carrier activity / SNARE complex / SNAP receptor activity / vesicle fusion / intra-Golgi vesicle-mediated transport / actomyosin / LGI-ADAM interactions / Golgi to plasma membrane protein transport / positive regulation of synaptic plasticity / positive regulation of ATP-dependent activity / ATP-dependent protein disaggregase activity / Golgi stack / response to cholesterol / neurotransmitter secretion / ATP-dependent protein binding / cytoplasmic side of membrane / insulin secretion / clathrin-dependent endocytosis / apical protein localization / regulation of synaptic vesicle cycle / clathrin-coated vesicle / syntaxin binding / protein localization to membrane / syntaxin-1 binding / Neutrophil degranulation / regulation of neuron projection development / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / endosomal transport / exocytosis / regulation of synapse assembly / myosin binding / SNARE complex assembly / neuron projection terminus / synaptic vesicle priming / response to gravity / synaptic vesicle exocytosis / positive regulation of receptor recycling / neurotransmitter transport / positive regulation of exocytosis
Similarity search - Function
Synaptobrevin/Vesicle-associated membrane protein / NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptobrevin signature. / Vesicle-fusing ATPase / : / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family ...Synaptobrevin/Vesicle-associated membrane protein / NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptobrevin signature. / Vesicle-fusing ATPase / : / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Synaptobrevin-like / Synaptobrevin / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / SNARE domain / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Tetratricopeptide-like helical domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 2
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 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
DepositionDec 5, 2014-
Header (metadata) releaseJan 28, 2015-
Map releaseJan 28, 2015-
UpdateFeb 11, 2015-
Current statusFeb 11, 2015Processing site: RCSB / Status: Released

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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
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j96
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6206.png

Downloads & links

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Map

FileDownload / File: emd_6206.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of 20S supercomplex, state I. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
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-4.49752235 - 12.11765003
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-4.49812.118-0.000

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Supplemental data

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Supplemental map: emd 6206 additional 1.map

Fileemd_6206_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 20S supercomplex consisting of truncated neuronal SNARE complex, ...

EntireName: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
Components
  • Sample: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
  • Protein or peptide: N-ethylmaleimide sensitive factor
  • Protein or peptide: alpha Soluble NSF Attachment Protein
  • Protein or peptide: Syntaxin-1A
  • Protein or peptide: Synaptobrevin-2
  • Protein or peptide: Synaptosomal-associated protein 25

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Supramolecule #1000: 20S supercomplex consisting of truncated neuronal SNARE complex, ...

SupramoleculeName: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
type: sample / ID: 1000
Oligomeric state: One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex
Number unique components: 5
Molecular weightTheoretical: 660 KDa

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Macromolecule #1: N-ethylmaleimide sensitive factor

MacromoleculeName: N-ethylmaleimide sensitive factor / type: protein_or_peptide / ID: 1 / Name.synonym: NSF / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Cricetulus griseus (Chinese hamster) / synonym: Chinese hamster
Molecular weightTheoretical: 83 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-RIL / Recombinant plasmid: pPROEX-1
SequenceUniProtKB: Vesicle-fusing ATPase

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Macromolecule #2: alpha Soluble NSF Attachment Protein

MacromoleculeName: alpha Soluble NSF Attachment Protein / type: protein_or_peptide / ID: 2 / Name.synonym: alpha-SNAP / Number of copies: 4 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightTheoretical: 33 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pJ414
SequenceUniProtKB: Alpha-soluble NSF attachment protein

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Macromolecule #3: Syntaxin-1A

MacromoleculeName: Syntaxin-1A / type: protein_or_peptide / ID: 3 / Name.synonym: Stx-1A / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightTheoretical: 8 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1
SequenceUniProtKB: Syntaxin-1A

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Macromolecule #4: Synaptobrevin-2

MacromoleculeName: Synaptobrevin-2 / type: protein_or_peptide / ID: 4 / Name.synonym: Syb-2, VAMP-2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightTheoretical: 8 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1
SequenceUniProtKB: Vesicle-associated membrane protein 2

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Macromolecule #5: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 5 / Name.synonym: SNAP-25 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightTheoretical: 16 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET-DUET-1
SequenceUniProtKB: Synaptosomal-associated protein 25

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration15 mg/mL
BufferpH: 8
Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
GridDetails: Holey carbon on top of 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 3.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
DateJan 28, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 44 e/Å2
Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: -2.8 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Details3D classification, refinement, and reconstruction were performed using RELION.
CTF correctionDetails: Each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 29717

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Atomic model buiding 1

Initial modelPDB ID:

1nsf


Chain - Chain ID: A
SoftwareName: Chimera, PHENIX
DetailsD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor
Output model

PDB-3j96:
Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State I)

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Atomic model buiding 2

Initial modelPDB ID:

1qcs


Chain - Chain ID: A
SoftwareName: Chimera, PHENIX
DetailsD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor
Output model

PDB-3j96:
Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State I)

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Atomic model buiding 3

Initial modelPDB ID:

1n7s


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D
SoftwareName: Chimera, PHENIX
DetailsD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor
Output model

PDB-3j96:
Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State I)

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