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- PDB-3j96: Structure of 20S supercomplex determined by single particle cryoe... -

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Basic information

Entry
Database: PDB / ID: 3j96
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy (State I)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25SNAP25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2Vesicle-associated membrane protein
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / vesicle trafficking
Function / homologyAAA+ lid domain / CDC48, N-terminal subdomain / SNAP-25 / Synaptobrevin / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) ...AAA+ lid domain / CDC48, N-terminal subdomain / SNAP-25 / Synaptobrevin / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / AAA+ ATPase domain / Synaptobrevin signature. / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2 / Syntaxin 1A / P-loop containing nucleoside triphosphate hydrolase / Synaptobrevin/Vesicle-associated membrane protein / Tetratricopeptide-like helical domain superfamily / SNARE / Aspartate decarboxylase-like domain superfamily / Syntaxin/epimorphin, conserved site / Syntaxin, N-terminal domain / CDC48, domain 2 / SNARE domain / AAA-protein family signature. / ATPase, AAA-type, core / Syntaxin / epimorphin family signature. / GABA synthesis, release, reuptake and degradation / Target SNARE coiled-coil homology domain / Clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / Retrograde transport at the Trans-Golgi-Network / Intra-Golgi traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / LGI-ADAM interactions / Other interleukin signaling / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / Clathrin derived vesicle budding / Acetylcholine Neurotransmitter Release Cycle / NSF attachment protein / Dopamine Neurotransmitter Release Cycle / Glutamate Neurotransmitter Release Cycle / COPII-mediated vesicle transport / Norepinephrine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / v-SNARE coiled-coil homology domain profile. / t-SNARE coiled-coil homology domain profile. / ATPase, AAA-type, conserved site / soluble NSF attachment protein activity / regulation of delayed rectifier potassium channel activity / SNARE complex disassembly / 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 complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II 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 / SNARE complex assembly / 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 / Golgi to plasma membrane protein transport / positive regulation of calcium ion-dependent exocytosis / calcium ion-regulated exocytosis of neurotransmitter / regulation of synapse assembly / vesicle docking / vesicle-fusing ATPase / protein localization to membrane / positive regulation of hormone secretion / chloride channel inhibitor activity / vesicle fusion / integral component of synaptic vesicle membrane / response to gravity / sleep / SNAP receptor activity / positive regulation of ATPase activity / regulation of neuron projection development / regulation of exocytosis / synaptic vesicle exocytosis / positive regulation of receptor recycling / SNARE complex / positive regulation of intracellular protein transport / membrane fusion
Function and homology information
Specimen sourceCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 7.6 Å resolution
AuthorsZhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Dec 5, 2014 / Release: Jan 28, 2015
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 28, 2015Structure modelrepositoryInitial release
1.1Feb 11, 2015Structure modelDatabase references
1.2Jul 18, 2018Structure modelData collectionem_image_scans / em_software_em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-6206
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
G: Alpha-soluble NSF attachment protein
H: Alpha-soluble NSF attachment protein
I: Alpha-soluble NSF attachment protein
J: Alpha-soluble NSF attachment protein
K: Vesicle-associated membrane protein 2
L: Syntaxin-1A
M: Synaptosomal-associated protein 25


Theoretical massNumber of molelcules
Total (without water)667,28213
Polyers667,28213
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide
Vesicle-fusing ATPase / / N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein NSF / N-ethylmaleimide sensitive factor


Mass: 82907.430 Da / Num. of mol.: 6 / Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase
#2: Protein/peptide
Alpha-soluble NSF attachment protein / SNAP-alpha / N-ethylmaleimide-sensitive factor attachment protein alpha / alpha-SNAP


Mass: 33377.793 Da / Num. of mol.: 4 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P54921
#3: Protein/peptide Vesicle-associated membrane protein 2 / Vesicle-associated membrane protein / VAMP-2 / Synaptobrevin-2


Mass: 7231.061 Da / Num. of mol.: 1 / Fragment: UNP residues 28-89 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli (E. coli) / References: UniProt: P63045
#4: Protein/peptide Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 7837.957 Da / Num. of mol.: 1 / Fragment: UNP residues 191-256 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#5: Protein/peptide Synaptosomal-associated protein 25 / SNAP25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 21256.855 Da / Num. of mol.: 1 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent ID
120S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)RIBOSOMEOne hexamer of NSF + four alpha-SNAP molecules + one SNARE complex0
2N-ethylmaleimide sensitive factor1
3alpha Soluble NSF Attachment Protein1
4Syntaxin-1A1
5Synaptobrevin-21
6Synaptosomal-associated protein 251
Molecular weightValue: 0.66 MDa / Experimental value: NO
Buffer solutionName: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
Details: 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
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Holey carbon on top of 400 mesh copper grid
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 %
Details: Blot for 3.5 seconds before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 3.5 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300 / Date: Jan 28, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: unspecified
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3RELION3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C1
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 29717 / Nominal pixel size: 2.4312 / Actual pixel size: 2.4312
Details: (Single particle details: 3D classification, refinement, and reconstruction were performed using RELION) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model buildingDetails: REFINEMENT PROTOCOL--flexible 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.
Ref protocol: FLEXIBLE FIT / Ref space: RECIPROCAL / Target criteria: R-factor
Atomic model building
IDPDB-IDPdb chain ID 3D fitting ID
11NSFA1
21QCSA1
31N7SA1
41N7SB1
51N7SC1
61N7SD1
RefineOverall SU ML: 1.53 / Sigma F: 0.5 / Overall phase error: 40.44 / Stereochemistry target values: ML
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å / Solvent model details: FLAT BULK SOLVENT MODEL
Displacement parametersB iso max: 55 Å2 / B iso mean: 500.074 Å2 / B iso min: 3 Å2
Least-squares processR factor R free: 0.3035 / R factor R work: 0.2997 / R factor obs: 0.2999 / Highest resolution: 7.601 Å / Lowest resolution: 311.194 Å / Number reflection R free: 7233 / Number reflection R work: 135735 / Number reflection obs: 142968 / Percent reflection R free: 5.06 / Percent reflection obs: 99.43
Refine hist #LASTHighest resolution: 7.601 Å / Lowest resolution: 311.194 Å
Number of atoms included #LASTProtein: 41139 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 41139
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00942091
ELECTRON MICROSCOPYf_angle_d1.59656840
ELECTRON MICROSCOPYf_chiral_restr0.0736546
ELECTRON MICROSCOPYf_plane_restr0.0067355
ELECTRON MICROSCOPYf_dihedral_angle_d15.90615528
Refine LS shell

Refine ID: ELECTRON MICROSCOPY / Total number of bins used: 30

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R workNumber reflection allPercent reflection obs
7.60140.52830.49957.687822844764704100.0
7.68780.51020.48207.778225249565208100.0
7.77820.50410.47367.873123242214453100.0
7.87310.45140.46457.972824047645004100.0
7.97280.44810.44408.077725242994551100.0
8.07770.43590.43378.188424048585098100.0
8.18840.42480.42088.305326443594623100.0
8.30530.38530.41068.429323145364767100.0
8.42930.43760.40598.561119247024894100.0
8.56110.43430.40028.701425243834635100.0
8.70140.43580.39738.851525245544806100.0
8.85150.39730.38349.012424046154855100.0
9.01240.38880.35419.185827644734749100.0
9.18580.33540.34199.373322845124740100.0
9.37330.35570.32119.577126446054869100.0
9.57710.34520.30779.799923946764915100.0
9.79990.30720.304210.04502404499473999.0
10.04500.28810.301510.31662394272451198.0
10.31660.29740.290610.62022274462468998.0
10.62020.28770.279010.96302724568484099.0
10.96300.28010.266611.35491984398459698.0
11.35490.28130.265611.80952344508474299.0
11.80950.28030.272412.34702134489470299.0
12.34700.29550.284812.99802064607481399.0
12.99800.37730.302613.81232864462474899.0
13.81230.32730.309614.87882384464470299.0
14.87880.33120.308116.37602644563482799.0
16.37600.35720.317918.74542514437468899.0
18.74540.33760.321423.61602504491474199.0
23.61600.16120.1774311.45502334526475999.0

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