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- EMDB-6207: Structure of 20S supercomplex determined by single particle cryoe... -

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Entry
Database: EMDB / ID: EMD-6207
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy, state II
Map data
Sample20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
  • N-ethylmaleimide sensitive factor
  • alpha Soluble NSF Attachment Protein
  • Syntaxin-1A
  • Synaptobrevin-2
  • Synaptosomal-associated protein 25SNAP25
Keywordsvesicle trafficking
Function / homology
Function and homology information


soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / anchored component of presynaptic membrane / zymogen granule membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / hormone secretion / eosinophil degranulation / growth hormone secretion ...soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / anchored component of presynaptic membrane / zymogen granule membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / hormone secretion / eosinophil degranulation / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex / neurotransmitter transport / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II 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 / regulated exocytosis / synaptic vesicle docking / protein-containing complex disassembly / intracellular organelle / storage vacuole / calcium ion-regulated exocytosis of neurotransmitter / neurotransmitter receptor internalization / regulation of synaptic vesicle recycling / vesicle docking / secretion by cell / positive regulation of calcium ion-dependent exocytosis / apical protein localization / protein localization to membrane / membrane fusion / integral component of synaptic vesicle membrane / positive regulation of neurotransmitter secretion / vesicle fusion / Golgi to plasma membrane protein transport / vesicle-fusing ATPase / regulation of vesicle-mediated transport / positive regulation of hormone secretion / SNARE complex / SNAP receptor activity / chloride channel inhibitor activity / SNARE complex assembly / calcium-ion regulated exocytosis / response to gravity / synaptic vesicle priming / regulation of exocytosis / positive regulation of ATPase activity / positive regulation of synaptic plasticity / positive regulation of intracellular protein transport / positive regulation of receptor recycling / regulation of synapse assembly / ATP-dependent protein binding / actomyosin / regulation of neuron projection development / sleep / syntaxin-1 binding / vacuolar membrane / synaptic vesicle exocytosis / neuron projection terminus / modulation of excitatory postsynaptic potential / myosin binding / neurotransmitter secretion / clathrin-coated vesicle / positive regulation of exocytosis / insulin secretion / syntaxin binding / protein sumoylation / voltage-gated potassium channel complex / exocytosis / ionotropic glutamate receptor binding / voltage-gated potassium channel activity / synaptic vesicle endocytosis / endomembrane system / associative learning / calcium channel inhibitor activity / long-term memory / positive regulation of insulin secretion / somatodendritic compartment / positive regulation of excitatory postsynaptic potential / SNARE binding / long-term synaptic potentiation / axonal growth cone / synaptic vesicle membrane / photoreceptor inner segment / axonogenesis / acrosomal vesicle / synaptic transmission, glutamatergic / locomotory behavior / vesicle-mediated transport / presynapse / filopodium / response to glucose / potassium ion transport / endosomal transport / ATPase activity, coupled / neuron differentiation
SNARE / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / CDC48, domain 2 / Syntaxin, N-terminal domain / Syntaxin/epimorphin, conserved site / Aspartate decarboxylase-like domain superfamily ...SNARE / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / CDC48, domain 2 / Syntaxin, N-terminal domain / Syntaxin/epimorphin, conserved site / Aspartate decarboxylase-like domain superfamily / Tetratricopeptide-like helical domain superfamily / Synaptobrevin/Vesicle-associated membrane protein / P-loop containing nucleoside triphosphate hydrolase / Syntaxin 1 / Vesicle-associated membrane protein 2 / CDC48 domain 2-like superfamily / Synaptosomal-associated protein 25 / NSF attachment protein / Vesicle-fusing ATPase / AAA ATPase, AAA+ lid domain / v-SNARE, coiled-coil homology domain / Target SNARE coiled-coil homology domain / SNAP-25 domain
Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 2
Biological speciesCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 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.
Validation ReportPDB-ID: 3j97

SummaryFull reportAbout validation report
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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  • Surface view with fitted model
  • Atomic models: : PDB-3j97
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6207.png

Downloads & links

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Map

FileDownload / File: emd_6207.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-4.15179205 - 11.32074547
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-4.15211.321-0.000

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Supplemental data

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Supplemental map: EMD-6207 State II 20S sharpened -601.map

FileEMD-6207_State_II_20S_sharpened_-601.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)
Number of components: 5
Oligomeric State: One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex
MassTheoretical: 660 kDa

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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: 8 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, Synaptobrevin-2

ProteinName: Synaptobrevin-2 / a.k.a: Syb-2, VAMP2 / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 8 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 2

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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: 16 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 AMPPNP, 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: Jan 28, 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: 21489
Details: 3D classification, refinement, and reconstruction were performed using RELION.
3D reconstructionSoftware: RELION / CTF correction: Each particle / Resolution: 7.8 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Chimera, PHENIX / Refinement protocol: flexible / Target criteria: R-factor / Refinement space: RECIPROCAL
Details: D2 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 ...Details: D2 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.
Input PDB model: 1NSF

1nsf


Chain ID: A
Modeling #2Software: Chimera, PHENIX / Refinement protocol: flexible / Target criteria: R-factor / Refinement space: RECIPROCAL
Details: D2 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 ...Details: D2 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.
Input PDB model: 1QCS

1qcs


Chain ID: A
Modeling #3Software: Chimera, PHENIX / Refinement protocol: flexible / Target criteria: R-factor / Refinement space: RECIPROCAL
Details: D2 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 ...Details: D2 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.
Input PDB model: 1N7S

1n7s


Chain ID: A, B, C, D
Output model

3j97

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