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Yorodumi- PDB-7udb: Cryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7udb | |||||||||||||||||||||
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Title | Cryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex class 2 | |||||||||||||||||||||
Components |
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Keywords | EXOCYTOSIS / Munc18 / syntaxin / synaptobrevin / SNARE / membrane fusion / neurotransmitter release | |||||||||||||||||||||
Function / homology | Function and homology information positive regulation of vesicle docking / regulation of acrosomal vesicle exocytosis / positive regulation of glutamate secretion, neurotransmission / negative regulation of SNARE complex assembly / regulation of vesicle fusion / developmental process involved in reproduction / axon target recognition / trans-Golgi Network Vesicle Budding / regulation of delayed rectifier potassium channel activity / myosin head/neck binding ...positive regulation of vesicle docking / regulation of acrosomal vesicle exocytosis / positive regulation of glutamate secretion, neurotransmission / negative regulation of SNARE complex assembly / regulation of vesicle fusion / developmental process involved in reproduction / axon target recognition / trans-Golgi Network Vesicle Budding / 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 / 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 / negative regulation of synaptic transmission, GABAergic / synaptic vesicle docking / storage vacuole / response to gravity / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / neuromuscular synaptic transmission / platelet degranulation / synaptic vesicle maturation / eosinophil degranulation / vesicle fusion / vesicle docking / positive regulation of calcium ion-dependent exocytosis / chloride channel inhibitor activity / secretion by cell / SNARE complex / SNAP receptor activity / presynaptic active zone cytoplasmic component / Cargo recognition for clathrin-mediated endocytosis / regulation of vesicle-mediated transport / Clathrin-mediated endocytosis / LGI-ADAM interactions / hormone secretion / calcium-ion regulated exocytosis / actomyosin / positive regulation of intracellular protein transport / positive regulation of mast cell degranulation / Golgi to plasma membrane protein transport / platelet alpha granule / regulation of exocytosis / neurotransmitter secretion / protein localization to membrane / ATP-dependent protein binding / presynaptic cytosol / neuron projection terminus / vesicle docking involved in exocytosis / neurotransmitter transport / regulation of synaptic vesicle recycling / parallel fiber to Purkinje cell synapse / insulin secretion / long-term synaptic depression / syntaxin-1 binding / SNARE complex assembly / positive regulation of neurotransmitter secretion / syntaxin binding / clathrin-coated vesicle / synaptic vesicle priming / myosin binding / exocytosis / synaptic vesicle exocytosis / positive regulation of exocytosis / modulation of excitatory postsynaptic potential / positive regulation of excitatory postsynaptic potential / phospholipase binding / protein sumoylation / synaptic vesicle endocytosis / calcium channel inhibitor activity / endomembrane system / negative regulation of protein-containing complex assembly / response to glucose / presynaptic active zone membrane / vesicle-mediated transport / phagocytic vesicle / SNARE binding / acrosomal vesicle / secretory granule / establishment of localization in cell / protein localization to plasma membrane / long-term synaptic potentiation / postsynaptic density membrane Similarity search - Function | |||||||||||||||||||||
Biological species | Rattus norvegicus (Norway rat) | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||||||||||||||
Authors | Rizo, J. / Bai, X. / Stepien, K.P. / Xu, J. / Zhang, X. | |||||||||||||||||||||
Funding support | United States, 6items
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Citation | Journal: Sci Adv / Year: 2022 Title: SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex. Authors: Karolina P Stepien / Junjie Xu / Xuewu Zhang / Xiao-Chen Bai / Josep Rizo / Abstract: Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal ...Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release. | |||||||||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7udb.cif.gz | 192.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7udb.ent.gz | 144.7 KB | Display | PDB format |
PDBx/mmJSON format | 7udb.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7udb_validation.pdf.gz | 913.1 KB | Display | wwPDB validaton report |
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Full document | 7udb_full_validation.pdf.gz | 918.3 KB | Display | |
Data in XML | 7udb_validation.xml.gz | 30.8 KB | Display | |
Data in CIF | 7udb_validation.cif.gz | 44.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ud/7udb ftp://data.pdbj.org/pub/pdb/validation_reports/ud/7udb | HTTPS FTP |
-Related structure data
Related structure data | 26455MC 7udcC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 68714.883 Da / Num. of mol.: 1 / Mutation: D326K Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stxbp1, Unc18a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P61765 |
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#2: Protein | Mass: 29221.541 Da / Num. of mol.: 1 / Mutation: C145A,L165A,E166A,L205C Source method: isolated from a genetically manipulated source Details: An 8 residue polyalanine stretch was modeled between I149 and A178 of chain B entity Syntaxin-1A. Side chains in this region were not visible and residue numbers are tentative. While ...Details: An 8 residue polyalanine stretch was modeled between I149 and A178 of chain B entity Syntaxin-1A. Side chains in this region were not visible and residue numbers are tentative. While processing in OneDep the polyalanine sequence was updated to 162-SEEAADML-169 Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P32851 |
#3: Protein | Mass: 6645.386 Da / Num. of mol.: 1 / Mutation: Q36C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P63045 |
Sequence details | An 8 residue polyalanine stretch was modeled between I149 and A178 of chain B entity Syntaxin-1A. ...An 8 residue polyalanine stretch was modeled between I149 and A178 of chain B entity Syntaxin-1A. Side chains in this region were not visible and residue numbers are tentative. While processing in OneDep the polyalanine sequence was updated to 162-SEEAADML-169 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2 Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7 |
Specimen | Conc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1600 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 7401 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 30 |
-Processing
Software | Name: PHENIX / Version: 1.19.1_4122: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 5819182 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 345463 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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