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Yorodumi- EMDB-6208: Structure of 20S supercomplex determined by single particle cryoe... -
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-Basic information
Entry | Database: EMDB / ID: EMD-6208 | |||||||||
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Title | Structure of 20S supercomplex determined by single particle cryoelectron microscopy, state IIIa | |||||||||
Map data | Map of 20S supercomplex, state IIIa. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN. | |||||||||
Sample |
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Keywords | 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 / extrinsic component of presynaptic membrane / synaptic vesicle fusion to presynaptic active zone 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 / positive regulation of norepinephrine secretion / regulation of synaptic vesicle priming / Glutamate Neurotransmitter Release Cycle / protein-containing complex disassembly / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / zymogen granule membrane / Acetylcholine Neurotransmitter Release Cycle / positive regulation of catecholamine secretion / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / regulated exocytosis / Dopamine Neurotransmitter Release Cycle / Golgi Associated Vesicle Biogenesis / regulation of establishment of protein localization / presynaptic dense core vesicle exocytosis / ribbon synapse / storage vacuole / synaptic vesicle docking / response to gravity / calcium ion-regulated exocytosis of neurotransmitter / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / eosinophil degranulation / positive regulation of calcium ion-dependent exocytosis / vesicle fusion / vesicle docking / chloride channel inhibitor activity / ATP-dependent protein disaggregase activity / Golgi to plasma membrane protein transport / SNARE complex / SNAP receptor activity / secretion by cell / regulation of vesicle-mediated transport / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / LGI-ADAM interactions / hormone secretion / calcium-ion regulated exocytosis / actomyosin / positive regulation of intracellular protein transport / neurotransmitter secretion / regulation of exocytosis / positive regulation of hormone secretion / apical protein localization / neurotransmitter receptor internalization / neuron projection terminus / neurotransmitter transport / ATP-dependent protein binding / protein localization to membrane / positive regulation of ATP-dependent activity / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / syntaxin-1 binding / insulin secretion / SNARE complex assembly / positive regulation of neurotransmitter secretion / synaptic vesicle priming / syntaxin binding / Neutrophil degranulation / clathrin-coated vesicle / regulation of synapse assembly / endosomal transport / myosin binding / positive regulation of receptor recycling / modulation of excitatory postsynaptic potential / regulation of neuron projection development / exocytosis / positive regulation of exocytosis / synaptic vesicle exocytosis / voltage-gated potassium channel activity / positive regulation of excitatory postsynaptic potential / associative learning / protein sumoylation / synaptic vesicle endocytosis / long-term memory / endomembrane system / calcium channel inhibitor activity / response to glucose / voltage-gated potassium channel complex / axonal growth cone / presynaptic active zone membrane / vesicle-mediated transport Similarity search - Function | |||||||||
Biological species | Cricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.4 Å | |||||||||
Authors | Zhao M / Wu S / Zhou Q / Vivona S / Cipriano DJ / Cheng Y / Brunger AT | |||||||||
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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6208.map.gz | 6 MB | EMDB map data format | |
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Header (meta data) | emd-6208-v30.xml emd-6208.xml | 17.4 KB 17.4 KB | Display Display | EMDB header |
Images | emd_6208.png | 109.5 KB | ||
Others | EMD-6208_State_IIIa_20S_sharpened_-612.map.gz | 7.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6208 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6208 | HTTPS FTP |
-Related structure data
Related structure data | 3j98MC 6204C 6205C 6206C 6207C 6209C 6210C 3j94C 3j95C 3j96C 3j97C 3j99C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6208.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map of 20S supercomplex, state IIIa. 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-6208 State IIIa 20S sharpened -612.map
File | EMD-6208_State_IIIa_20S_sharpened_-612.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-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: Cricetulus griseus (Chinese hamster) / synonym: Chinese hamster |
Molecular weight | Theoretical: 83 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21-DE3-RIL / Recombinant plasmid: pROEX-1 |
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: Rattus norvegicus (Norway rat) / synonym: Rat |
Molecular weight | Theoretical: 33 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pJ414 |
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: Rattus norvegicus (Norway rat) / synonym: Rat |
Molecular weight | Theoretical: 8 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1 |
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: Rattus norvegicus (Norway rat) / synonym: Rat |
Molecular weight | Theoretical: 8 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1 |
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: Rattus norvegicus (Norway rat) / synonym: Rat |
Molecular weight | Theoretical: 16 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET-DUET-1 |
Sequence | UniProtKB: Synaptosomal-associated protein 25 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-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. |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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 |
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. |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each particle |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 15249 |
Details | 3D classification, refinement, and reconstruction were performed using RELION. |
-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-3j98: |
-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-3j98: |
-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-3j98: |