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Yorodumi- PDB-3j99: Structure of 20S supercomplex determined by single particle cryoe... -
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-Basic information
Entry | Database: PDB / ID: 3j99 | ||||||
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Title | Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State IIIb) | ||||||
Components |
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Keywords | HYDROLASE / vesicle trafficking | ||||||
Function / homology | Function and homology information soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / trans-Golgi Network Vesicle Budding / BLOC-1 complex / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding ...soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / trans-Golgi Network Vesicle Budding / BLOC-1 complex / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / extrinsic component of presynaptic membrane / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / positive regulation of norepinephrine secretion / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / positive regulation of catecholamine secretion / Acetylcholine Neurotransmitter Release Cycle / Lysosome Vesicle Biogenesis / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / zymogen granule membrane / regulated exocytosis / ribbon synapse / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / storage vacuole / regulation of establishment of protein localization / response to gravity / protein-containing complex disassembly / vesicle-mediated transport in synapse / positive regulation of calcium ion-dependent exocytosis / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / calcium ion-regulated exocytosis of neurotransmitter / vesicle fusion / eosinophil degranulation / vesicle docking / secretion by cell / ATP-dependent protein disaggregase activity / SNARE complex / chloride channel inhibitor activity / SNAP receptor activity / regulation of exocytosis / regulation of vesicle-mediated transport / Cargo recognition for clathrin-mediated endocytosis / LGI-ADAM interactions / Clathrin-mediated endocytosis / calcium-ion regulated exocytosis / intra-Golgi vesicle-mediated transport / hormone secretion / actomyosin / positive regulation of intracellular protein transport / Golgi to plasma membrane protein transport / apical protein localization / Golgi stack / positive regulation of hormone secretion / neurotransmitter secretion / neurotransmitter receptor internalization / protein localization to membrane / ATP-dependent protein binding / neuron projection terminus / positive regulation of ATP-dependent activity / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / insulin secretion / syntaxin binding / neurotransmitter transport / syntaxin-1 binding / clathrin-coated vesicle / SNARE complex assembly / positive regulation of neurotransmitter secretion / Neutrophil degranulation / endosomal transport / synaptic vesicle priming / regulation of synapse assembly / postsynaptic cytosol / myosin binding / positive regulation of receptor recycling / regulation of neuron projection development / modulation of excitatory postsynaptic potential / exocytosis / associative learning / synaptic vesicle exocytosis / positive regulation of exocytosis / protein sumoylation / voltage-gated potassium channel activity / positive regulation of excitatory postsynaptic potential / synaptic vesicle endocytosis / calcium channel inhibitor activity / endomembrane system / response to glucose / long-term memory Similarity search - Function | ||||||
Biological species | Cricetulus griseus (Chinese hamster) Rattus norvegicus (Norway rat) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 Å | ||||||
Authors | Zhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 3j99.cif.gz | 882.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb3j99.ent.gz | 695.6 KB | Display | PDB format |
PDBx/mmJSON format | 3j99.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 3j99_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 3j99_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 3j99_validation.xml.gz | 158.9 KB | Display | |
Data in CIF | 3j99_validation.cif.gz | 239.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/j9/3j99 ftp://data.pdbj.org/pub/pdb/validation_reports/j9/3j99 | HTTPS FTP |
-Related structure data
Related structure data | 6209MC 6204C 6205C 6206C 6207C 6208C 6210C 3j94C 3j95C 3j96C 3j97C 3j98C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 82907.430 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase #2: Protein | Mass: 33377.793 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P54921 #3: Protein | | Mass: 7231.061 Da / Num. of mol.: 1 / Fragment: UNP residues 28-89 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli (E. coli) / References: UniProt: P63045 #4: Protein | | Mass: 7837.957 Da / Num. of mol.: 1 / Fragment: UNP residues 191-256 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851 #5: Protein | | Mass: 21256.855 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight | Value: 0.66 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Buffer solution | Name: 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 Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40 | |||||||||||||||||||||||||||||||||||
Specimen | Conc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Details: Holey carbon on top of 400 mesh copper grid | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 77 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. |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 / Date: Jan 28, 2014 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm |
Specimen holder | Specimen holder model: OTHER / Specimen holder type: unspecified |
Image recording | Electron dose: 44 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Relative weight: 1 |
-Processing
EM software |
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CTF correction | Details: Each particle | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14991 / Nominal pixel size: 2.4312 Å / Actual pixel size: 2.4312 Å Details: (Single particle details: 3D classification, refinement, and reconstruction were performed using RELION) (Single particle--Applied symmetry: C1) Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: R-factor Details: 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. ...Details: 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. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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Refinement | Resolution: 8.2→8.2 Å / SU ML: 1.57 / σ(F): 0.79 / Phase error: 39.71 / Stereochemistry target values: ML
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 50 Å2 / Biso mean: 50 Å2 / Biso min: 50 Å2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 8.201→311.194 Å
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Refine LS restraints |
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LS refinement shell | Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 30
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