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- PDB-3j97: Structure of 20S supercomplex determined by single particle cryoe... -
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Basic information
Entry | Database: PDB / ID: 3j97 | ||||||
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Title | Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State II) | ||||||
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![]() | HYDROLASE / vesicle trafficking | ||||||
Function / homology | ![]() 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 / extrinsic component of presynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / Lysosome Vesicle Biogenesis / synaptic vesicle fusion to presynaptic active zone membrane / regulation of synaptic vesicle priming / Glutamate Neurotransmitter Release Cycle / positive regulation of norepinephrine secretion / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / protein-containing complex disassembly / Acetylcholine Neurotransmitter Release Cycle / positive regulation of catecholamine secretion / zymogen granule membrane / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / Golgi Associated Vesicle Biogenesis / regulated exocytosis / presynaptic dense core vesicle exocytosis / ribbon synapse / synaptic vesicle docking / regulation of establishment of protein localization / storage vacuole / response to gravity / calcium ion-regulated exocytosis of neurotransmitter / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / eosinophil degranulation / vesicle fusion / vesicle docking / positive regulation of calcium ion-dependent exocytosis / chloride channel inhibitor activity / secretion by cell / ATP-dependent protein disaggregase activity / SNARE complex / SNAP receptor activity / Cargo recognition for clathrin-mediated endocytosis / regulation of vesicle-mediated transport / Clathrin-mediated endocytosis / LGI-ADAM interactions / hormone secretion / calcium-ion regulated exocytosis / actomyosin / apical protein localization / positive regulation of intracellular protein transport / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / regulation of exocytosis / positive regulation of hormone secretion / neurotransmitter secretion / Golgi stack / neurotransmitter receptor internalization / protein localization to membrane / ATP-dependent protein binding / neuron projection terminus / positive regulation of ATP-dependent activity / vesicle-fusing ATPase / neurotransmitter transport / regulation of synaptic vesicle recycling / insulin secretion / syntaxin-1 binding / SNARE complex assembly / positive regulation of neurotransmitter secretion / syntaxin binding / vacuolar membrane / clathrin-coated vesicle / synaptic vesicle priming / Neutrophil degranulation / regulation of synapse assembly / regulation of neuron projection development / endosomal transport / myosin binding / positive regulation of receptor recycling / exocytosis / voltage-gated potassium channel activity / synaptic vesicle exocytosis / positive regulation of exocytosis / modulation of excitatory postsynaptic potential / associative learning / positive regulation of excitatory postsynaptic potential / protein sumoylation / synaptic vesicle endocytosis / calcium channel inhibitor activity / endomembrane system / long-term memory / axonal growth cone / response to glucose Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.8 Å | ||||||
![]() | Zhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T. | ||||||
![]() | ![]() 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
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 884 KB | Display | ![]() |
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PDB format | ![]() | 697.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.3 MB | Display | |
Data in XML | ![]() | 161.8 KB | Display | |
Data in CIF | ![]() | 242.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6207MC ![]() 6204C ![]() 6205C ![]() 6206C ![]() 6208C ![]() 6209C ![]() 6210C ![]() 3j94C ![]() 3j95C ![]() 3j96C ![]() 3j98C ![]() 3j99C C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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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.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 33377.793 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #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.) ![]() ![]() ![]() ![]() #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.) ![]() ![]() ![]() ![]() #5: Protein | | Mass: 22576.363 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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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 |
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Sample preparation
Component |
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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: 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 |
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Microscopy | Model: FEI POLARA 300 / Date: Jan 28, 2014 |
Electron gun | Electron source: ![]() |
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 |
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Processing
EM software |
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CTF correction | Details: Each particle | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21489 / 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: 7.8→7.8 Å / SU ML: 1.51 / σ(F): 0.19 / Phase error: 40.21 / 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: 7.804→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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