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- EMDB-6206: Structure of 20S supercomplex determined by single particle cryoe... -
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Basic information
Entry | Database: EMDB / ID: EMD-6206 | |||||||||
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Title | Structure of 20S supercomplex determined by single particle cryoelectron microscopy, state I | |||||||||
![]() | Map of 20S supercomplex, state I. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN. | |||||||||
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![]() | 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 / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / Lysosome Vesicle Biogenesis / 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 / regulation of synaptic vesicle priming / 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 / positive regulation of calcium ion-dependent exocytosis / vesicle fusion / eosinophil degranulation / vesicle docking / 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 / regulation of exocytosis / LGI-ADAM interactions / hormone secretion / calcium-ion regulated exocytosis / actomyosin / positive regulation of intracellular protein transport / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / positive regulation of hormone secretion / apical protein localization / neurotransmitter secretion / Golgi stack / neurotransmitter receptor internalization / ATP-dependent protein binding / protein localization to membrane / neuron projection terminus / positive regulation of ATP-dependent activity / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / neurotransmitter transport / insulin secretion / syntaxin binding / syntaxin-1 binding / SNARE complex assembly / positive regulation of neurotransmitter secretion / clathrin-coated vesicle / Neutrophil degranulation / synaptic vesicle priming / endosomal transport / regulation of synapse assembly / postsynaptic cytosol / myosin binding / positive regulation of receptor recycling / regulation of neuron projection development / exocytosis / voltage-gated potassium channel activity / positive regulation of exocytosis / synaptic vesicle exocytosis / modulation of excitatory postsynaptic potential / associative learning / positive regulation of excitatory postsynaptic potential / protein sumoylation / synaptic vesicle endocytosis / endomembrane system / calcium channel inhibitor activity / long-term memory / response to glucose / axonal growth cone Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.6 Å | |||||||||
![]() | Zhao M / Wu S / Zhou Q / Vivona S / Cipriano DJ / Cheng Y / Brunger AT | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 17.4 KB 17.4 KB | Display Display | ![]() |
Images | ![]() | 119.6 KB | ||
Others | ![]() | 7.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j96MC ![]() 6204C ![]() 6205C ![]() 6207C ![]() 6208C ![]() 6209C ![]() 6210C ![]() 3j94C ![]() 3j95C ![]() 3j97C ![]() 3j98C ![]() 3j99C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Map of 20S supercomplex, state I. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.4312 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Supplemental map: emd 6206 additional 1.map
File | emd_6206_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : 20S supercomplex consisting of truncated neuronal SNARE complex, ...
Entire | Name: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF) |
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Components |
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-Supramolecule #1000: 20S supercomplex consisting of truncated neuronal SNARE complex, ...
Supramolecule | Name: 20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF) type: sample / ID: 1000 Oligomeric state: One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex Number unique components: 5 |
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Molecular weight | Theoretical: 660 KDa |
-Macromolecule #1: N-ethylmaleimide sensitive factor
Macromolecule | Name: N-ethylmaleimide sensitive factor / type: protein_or_peptide / ID: 1 / Name.synonym: NSF / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 83 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Vesicle-fusing ATPase |
-Macromolecule #2: alpha Soluble NSF Attachment Protein
Macromolecule | Name: alpha Soluble NSF Attachment Protein / type: protein_or_peptide / ID: 2 / Name.synonym: alpha-SNAP / Number of copies: 4 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 33 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Alpha-soluble NSF attachment protein |
-Macromolecule #3: Syntaxin-1A
Macromolecule | Name: Syntaxin-1A / type: protein_or_peptide / ID: 3 / Name.synonym: Stx-1A / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 8 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Syntaxin-1A |
-Macromolecule #4: Synaptobrevin-2
Macromolecule | Name: Synaptobrevin-2 / type: protein_or_peptide / ID: 4 / Name.synonym: Syb-2, VAMP-2 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 8 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Vesicle-associated membrane protein 2 |
-Macromolecule #5: Synaptosomal-associated protein 25
Macromolecule | Name: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 5 / Name.synonym: SNAP-25 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 16 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Synaptosomal-associated protein 25 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 15 mg/mL |
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Buffer | 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 |
Grid | Details: Holey carbon on top of 400 mesh copper grid |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 3.5 seconds before plunging. |
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Electron microscopy
Microscope | FEI POLARA 300 |
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Date | Jan 28, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 44 e/Å2 Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: -2.8 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 31000 |
Sample stage | Specimen holder model: OTHER |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Details | 3D classification, refinement, and reconstruction were performed using RELION. |
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CTF correction | Details: Each particle |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 29717 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, PHENIX |
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. |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor |
Output model | ![]() PDB-3j96: |
-Atomic model buiding 2
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, PHENIX |
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. |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor |
Output model | ![]() PDB-3j96: |
-Atomic model buiding 3
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D |
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Software | Name: Chimera, PHENIX |
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. |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor |
Output model | ![]() PDB-3j96: |