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Yorodumi- EMDB-6210: Structure of 20S supercomplex with V7-SNARE determined by single ... -
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-Basic information
Entry | Database: EMDB / ID: EMD-6210 | |||||||||
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Title | Structure of 20S supercomplex with V7-SNARE determined by single particle cryoelectron microscopy | |||||||||
Map data | Map of 20S supercomplex with V7-SNARE. 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 regulation of protein targeting to vacuolar membrane / positive regulation of histamine secretion by mast cell / : / soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / triglyceride transport / natural killer cell degranulation / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport ...regulation of protein targeting to vacuolar membrane / positive regulation of histamine secretion by mast cell / : / soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / triglyceride transport / natural killer cell degranulation / 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 / vesicle fusion with Golgi apparatus / 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 / neutrophil degranulation / 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 / vesicle transport along microtubule / SNARE complex / SNAP receptor activity / Cargo recognition for clathrin-mediated endocytosis / regulation of vesicle-mediated transport / regulation of exocytosis / Clathrin-mediated endocytosis / 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 / platelet alpha granule / 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 / positive regulation of dendrite morphogenesis / 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 / azurophil granule membrane / regulation of synapse assembly / postsynaptic cytosol / myosin binding / endosome to lysosome transport / phagocytosis, engulfment / positive regulation of receptor recycling / regulation of neuron projection development / exocytosis Similarity search - Function | |||||||||
Biological species | Cricetulus griseus (Chinese hamster) / Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.0 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_6210.map.gz | 6 MB | EMDB map data format | |
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Header (meta data) | emd-6210-v30.xml emd-6210.xml | 14 KB 14 KB | Display Display | EMDB header |
Images | emd_6210.png | 91.4 KB | ||
Others | emd_6210_additional_1.map.gz | 7.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6210 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6210 | HTTPS FTP |
-Validation report
Summary document | emd_6210_validation.pdf.gz | 78.9 KB | Display | EMDB validaton report |
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Full document | emd_6210_full_validation.pdf.gz | 78 KB | Display | |
Data in XML | emd_6210_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6210 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6210 | HTTPS FTP |
-Related structure data
Related structure data | 6204C 6205C 6206C 6207C 6208C 6209C 3j94C 3j95C 3j96C 3j97C 3j98C 3j99C 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_6210.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 with V7-SNARE. 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 6210 additional 1.map
File | emd_6210_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, ...
Entire | Name: 20S supercomplex consisting of VAMP-7 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 VAMP-7 SNARE complex, alpha-SNAP, ...
Supramolecule | Name: 20S supercomplex consisting of VAMP-7 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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-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: 30 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1 |
Sequence | UniProtKB: Syntaxin-1A |
-Macromolecule #4: Vesicle-associated membrane protein 7
Macromolecule | Name: Vesicle-associated membrane protein 7 / type: protein_or_peptide / ID: 4 / Name.synonym: VAMP-7 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) / synonym: Rat |
Molecular weight | Theoretical: 22 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pACYC-DUET-1 |
Sequence | UniProtKB: Vesicle-associated membrane protein 7 |
-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: 25 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 ATP, 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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Date | May 12, 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: FIELD EMISSION GUN |
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 |
-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: 8.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 32100 |