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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 dataMap of 20S supercomplex with V7-SNARE. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
Sample
  • Sample: 20S supercomplex consisting of VAMP-7 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: Vesicle-associated membrane protein 7
  • Protein or peptide: Synaptosomal-associated protein 25
Keywordsvesicle trafficking
Function / homology
Function and homology information


Interleukin-12 signaling / regulation of protein targeting to vacuolar membrane / positive regulation of histamine secretion by mast cell / neutrophil degranulation / triglyceride transport / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / natural killer cell degranulation / trans-Golgi Network Vesicle Budding / regulation of delayed rectifier potassium channel activity / vesicle fusion with Golgi apparatus ...Interleukin-12 signaling / regulation of protein targeting to vacuolar membrane / positive regulation of histamine secretion by mast cell / neutrophil degranulation / triglyceride transport / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / natural killer cell degranulation / trans-Golgi Network Vesicle Budding / regulation of delayed rectifier potassium channel activity / vesicle fusion with Golgi apparatus / 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 / BLOC-1 complex / Lysosome Vesicle Biogenesis / myosin head/neck binding / zymogen granule membrane / storage vacuole / synaptic vesicle fusion to presynaptic active zone membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / extrinsic component of presynaptic membrane / calcium ion-regulated exocytosis of neurotransmitter / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / COPII-mediated vesicle transport / GABA synthesis, release, reuptake and degradation / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / Dopamine Neurotransmitter Release Cycle / synaptic vesicle docking / eosinophil degranulation / SNARE complex disassembly / regulated exocytosis / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / vesicle-mediated transport in synapse / protein-containing complex disassembly / positive regulation of intracellular protein transport / regulation of establishment of protein localization / positive regulation of calcium ion-dependent exocytosis / ribbon synapse / regulation of vesicle-mediated transport / vesicle docking / Cargo recognition for clathrin-mediated endocytosis / regulation of exocytosis / secretion by cell / chloride channel inhibitor activity / Clathrin-mediated endocytosis / platelet alpha granule / SNAP receptor activity / SNARE complex / calcium-ion regulated exocytosis / vesicle fusion / positive regulation of dendrite morphogenesis / ATP-dependent protein disaggregase activity / hormone secretion / actomyosin / LGI-ADAM interactions / positive regulation of ATP-dependent activity / positive regulation of hormone secretion / intra-Golgi vesicle-mediated transport / neuron projection terminus / Golgi to plasma membrane protein transport / ATP-dependent protein binding / Golgi stack / vesicle transport along microtubule / neurotransmitter secretion / protein localization to membrane / clathrin-coated vesicle / apical protein localization / syntaxin binding / regulation of synaptic vesicle recycling / vesicle-fusing ATPase / insulin secretion / syntaxin-1 binding / endosomal transport / azurophil granule membrane / Neutrophil degranulation / phagocytosis, engulfment / SNARE complex assembly / positive regulation of neurotransmitter secretion / endosome to lysosome transport / neurotransmitter transport / regulation of synapse assembly / synaptic vesicle priming / myosin binding / response to gravity / regulation of neuron projection development / pseudopodium / positive regulation of receptor recycling / exocytosis
Similarity search - Function
: / Longin domain profile. / Longin domain / Regulated-SNARE-like domain / Regulated-SNARE-like domain / Synaptobrevin/Vesicle-associated membrane protein / NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptobrevin signature. / : ...: / Longin domain profile. / Longin domain / Regulated-SNARE-like domain / Regulated-SNARE-like domain / Synaptobrevin/Vesicle-associated membrane protein / NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptobrevin signature. / : / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / Synaptobrevin-like / SNAP-25 family / Synaptobrevin / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / Vesicle-fusing ATPase / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Longin-like domain superfamily / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / 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 / Vesicle-associated membrane protein 7
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 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
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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)
AnnotationMap of 20S supercomplex with V7-SNARE. 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-4.03881359 - 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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Supplemental map: emd 6210 additional 1.map

Fileemd_6210_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 VAMP-7 SNARE complex, alpha-SNAP, ...

EntireName: 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
Components
  • Sample: 20S supercomplex consisting of VAMP-7 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: Vesicle-associated membrane protein 7
  • Protein or peptide: Synaptosomal-associated protein 25

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Supramolecule #1000: 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, ...

SupramoleculeName: 20S supercomplex consisting of VAMP-7 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

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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: pROEX-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: 30 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: Vesicle-associated membrane protein 7

MacromoleculeName: Vesicle-associated membrane protein 7 / type: protein_or_peptide / ID: 4 / Name.synonym: VAMP-7 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightTheoretical: 22 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1
SequenceUniProtKB: Vesicle-associated membrane protein 7

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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: 25 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 ATP, 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
DateMay 12, 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: 8.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 32100

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