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- EMDB-6208: Structure of 20S supercomplex determined by single particle cryoe... -

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Entry
Database: EMDB / ID: 6208
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy, state IIIa
Map dataMap of 20S supercomplex, state IIIa. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
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 / homologyv-SNARE coiled-coil homology domain profile. / NSF attachment protein / Syntaxin, N-terminal domain / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / Synaptobrevin / SNAP-25 ...v-SNARE coiled-coil homology domain profile. / NSF attachment protein / Syntaxin, N-terminal domain / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / Synaptobrevin / SNAP-25 / Intra-Golgi traffic / COPI-dependent Golgi-to-ER retrograde traffic / Target SNARE coiled-coil homology domain / Aspartate decarboxylase-like domain superfamily / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / Glutamate Neurotransmitter Release Cycle / Dopamine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Clathrin derived vesicle budding / Lysosome Vesicle Biogenesis / Serotonin Neurotransmitter Release Cycle / Golgi Associated Vesicle Biogenesis / Other interleukin signaling / Syntaxin/epimorphin, conserved site / SNARE / LGI-ADAM interactions / Tetratricopeptide-like helical domain superfamily / t-SNARE coiled-coil homology domain profile. / Syntaxin / epimorphin family signature. / AAA-protein family signature. / Synaptobrevin signature. / SNARE domain / Cell division protein 48 (CDC48), domain 2 / GABA synthesis, release, reuptake and degradation / Cell division protein 48 (CDC48), N-terminal domain / Clathrin-mediated endocytosis / Synaptobrevin / SNAP-25 family / Syntaxin / Cargo recognition for clathrin-mediated endocytosis / Retrograde transport at the Trans-Golgi-Network / ATPase family associated with various cellular activities (AAA) / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2 / Syntaxin 1A / P-loop containing nucleoside triphosphate hydrolase / Synaptobrevin/Vesicle-associated membrane protein / COPI-mediated anterograde transport / soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / storage vacuole / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / myosin head/neck binding / anchored component of presynaptic membrane / regulation of synaptic vesicle priming / zymogen granule membrane / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / neurotransmitter transport / 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 / synaptic vesicle docking / regulation of synaptic vesicle recycling / synaptic vesicle priming / intracellular organelle / positive regulation of neurotransmitter secretion / apical protein localization / protein-containing complex disassembly / calcium ion-regulated exocytosis of neurotransmitter / positive regulation of calcium ion-dependent exocytosis / regulation of synapse assembly / vesicle docking / SNARE complex assembly / vesicle-fusing ATPase / positive regulation of intracellular protein transport / protein localization to membrane / Golgi to plasma membrane protein transport / vesicle fusion / response to gravity / integral component of synaptic vesicle membrane / chloride channel inhibitor activity / positive regulation of hormone secretion / SNAP receptor activity / sleep / regulation of exocytosis / positive regulation of ATPase activity / SNARE complex / positive regulation of receptor recycling / membrane fusion / regulation of neuron projection development / synaptic vesicle exocytosis / neuron projection terminus
Function and homology information
SourceCricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / 8.4 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
Validation ReportPDB-ID: 3j98

SummaryFull reportAbout validation report
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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  • Surface view with fitted model
  • Atomic models: : PDB-3j98
  • 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_6208.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-4.68995571 - 11.37624359
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-4.69011.376-0.000

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Supplemental data

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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, VAMP-2 / 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: 15249
Details: 3D classification, refinement, and reconstruction were performed using RELION.
3D reconstructionSoftware: RELION / CTF correction: Each particle / Resolution: 8.4 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 was from crystal structure 1N7S.
Input PDB model: 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 was from crystal structure 1N7S.
Input PDB model: 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 was from crystal structure 1N7S.
Input PDB model: 1N7S
Chain ID: A, B, C, D
Output model

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