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- PDB-7udb: Cryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex ... -

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Basic information

Entry
Database: PDB / ID: 7udb
TitleCryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex class 2
Components
  • Synaptobrevin-2
  • Syntaxin-1A
  • Syntaxin-binding protein 1
KeywordsEXOCYTOSIS / 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 / developmental process involved in reproduction / axon target recognition / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / trans-Golgi Network Vesicle Budding / regulation of vesicle fusion / regulation of delayed rectifier potassium channel activity ...positive regulation of vesicle docking / regulation of acrosomal vesicle exocytosis / positive regulation of glutamate secretion, neurotransmission / negative regulation of SNARE complex assembly / developmental process involved in reproduction / axon target recognition / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / trans-Golgi Network Vesicle Budding / regulation of vesicle fusion / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / Other interleukin signaling / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Lysosome Vesicle Biogenesis / regulated exocytosis / Dopamine Neurotransmitter Release Cycle / extrinsic component of presynaptic membrane / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / synaptic vesicle docking / negative regulation of synaptic transmission, GABAergic / zymogen granule membrane / regulation of synaptic vesicle priming / Golgi Associated Vesicle Biogenesis / storage vacuole / platelet degranulation / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / response to gravity / vesicle-mediated transport in synapse / synaptic vesicle maturation / positive regulation of calcium ion-dependent exocytosis / vesicle docking / eosinophil degranulation / presynaptic active zone cytoplasmic component / regulation of exocytosis / secretion by cell / SNAP receptor activity / SNARE complex / chloride channel inhibitor activity / vesicle fusion / positive regulation of mast cell degranulation / regulation of vesicle-mediated transport / calcium-ion regulated exocytosis / Cargo recognition for clathrin-mediated endocytosis / LGI-ADAM interactions / Clathrin-mediated endocytosis / actomyosin / positive regulation of intracellular protein transport / hormone secretion / neuromuscular synaptic transmission / platelet alpha granule / Golgi to plasma membrane protein transport / neurotransmitter secretion / ATP-dependent protein binding / neuron projection terminus / vesicle docking involved in exocytosis / protein localization to membrane / syntaxin binding / regulation of synaptic vesicle recycling / syntaxin-1 binding / long-term synaptic depression / clathrin-coated vesicle / insulin secretion / SNARE complex assembly / positive regulation of neurotransmitter secretion / neurotransmitter transport / synaptic vesicle priming / myosin binding / parallel fiber to Purkinje cell synapse / exocytosis / phospholipase binding / modulation of excitatory postsynaptic potential / positive regulation of exocytosis / synaptic vesicle exocytosis / protein sumoylation / postsynaptic cytosol / synaptic vesicle endocytosis / positive regulation of excitatory postsynaptic potential / negative regulation of protein-containing complex assembly / presynaptic cytosol / calcium channel inhibitor activity / response to glucose / vesicle-mediated transport / phagocytic vesicle / presynaptic active zone membrane / endomembrane system / cytoplasmic vesicle membrane / SNARE binding / acrosomal vesicle / secretory granule / protein localization to plasma membrane / establishment of localization in cell
Similarity search - Function
Syntaxin Binding Protein 1; Chain A, domain 2 / Sec1/Munc18 (SM) protein, domain 3a / Sec1/Munc18 (SM) protein, domain 1 / Sec1-like, domain 1 / Sec1-like protein / Sec1-like, domain 2 / Sec1-like superfamily / Sec1-like, domain 3a / Sec1 family / Synaptobrevin/Vesicle-associated membrane protein ...Syntaxin Binding Protein 1; Chain A, domain 2 / Sec1/Munc18 (SM) protein, domain 3a / Sec1/Munc18 (SM) protein, domain 1 / Sec1-like, domain 1 / Sec1-like protein / Sec1-like, domain 2 / Sec1-like superfamily / Sec1-like, domain 3a / Sec1 family / Synaptobrevin/Vesicle-associated membrane protein / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Synaptobrevin-like / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / Synaptobrevin / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / SNARE / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Alpha-Beta Complex / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Syntaxin-1A / Syntaxin-binding protein 1 / Vesicle-associated membrane protein 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRizo, J. / Bai, X. / Stepien, K.P. / Xu, J. / Zhang, X.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35 NS097333 United States
Robert A. Welch FoundationI-1304 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD030312-01 United States
Robert A. Welch FoundationI-1441 United States
Robert A. Welch FoundationI-1702 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130289 United States
CitationJournal: 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
DepositionMar 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
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Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
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Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Syntaxin-binding protein 1
B: Syntaxin-1A
C: Synaptobrevin-2


Theoretical massNumber of molelcules
Total (without water)104,5823
Polymers104,5823
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Syntaxin-binding protein 1 / N-Sec1 / Protein unc-18 homolog 1 / Unc18-1 / Protein unc-18 homolog A / Unc-18A / p67 / rbSec1


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
#2: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


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 Synaptobrevin-2 / VAMP-2 / Vesicle-associated membrane protein 2


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
Has protein modificationY
Sequence detailsAn 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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1600 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
8PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5819182
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 345463 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026563
ELECTRON MICROSCOPYf_angle_d0.4858828
ELECTRON MICROSCOPYf_dihedral_angle_d3.482870
ELECTRON MICROSCOPYf_chiral_restr0.0381008
ELECTRON MICROSCOPYf_plane_restr0.0041132

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