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- EMDB-26455: Cryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26455
TitleCryo-EM structure of a synaptobrevin-Munc18-1-syntaxin-1 complex class 2
Map dataCryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1
Sample
  • Complex: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2
    • Protein or peptide: Syntaxin-binding protein 1
    • Protein or peptide: Syntaxin-1A
    • Protein or peptide: Synaptobrevin-2
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
Sec1-like, domain 1 / Sec1-like, domain 2 / Sec1-like, domain 3a / Sec1-like protein / Sec1-like superfamily / Sec1 family / Synaptobrevin/Vesicle-associated membrane protein / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain ...Sec1-like, domain 1 / Sec1-like, domain 2 / Sec1-like, domain 3a / Sec1-like protein / Sec1-like superfamily / Sec1 family / Synaptobrevin/Vesicle-associated membrane protein / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / SNARE domain / Synaptobrevin-like / Syntaxin/epimorphin, conserved site / Synaptobrevin / Syntaxin / epimorphin family signature. / 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
Similarity search - Domain/homology
Syntaxin-1A / Syntaxin-binding protein 1 / Vesicle-associated membrane protein 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRizo J / Bai X / Stepien KP / Xu J / Zhang X
Funding support United States, 6 items
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, 2022-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateJul 6, 2022-
Current statusJul 6, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26455.png

Downloads & links

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Map

FileDownload / File: emd_26455.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.34721613 - 0.56915826
Average (Standard dev.)0.0004786197 (±0.013151482)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 194.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1, unfiltered...

Fileemd_26455_half_map_1.map
AnnotationCryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1, unfiltered half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1, unfiltered...

Fileemd_26455_half_map_2.map
AnnotationCryo-EM map of synaptobrevin-Munc18-1-syntaxin-1 complex, conformation 1, unfiltered half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE moti...

EntireName: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2
Components
  • Complex: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2
    • Protein or peptide: Syntaxin-binding protein 1
    • Protein or peptide: Syntaxin-1A
    • Protein or peptide: Synaptobrevin-2

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Supramolecule #1: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE moti...

SupramoleculeName: ternary complex of Munc18-1 bound to syntaxin-1A and a SNARE motif of synaptobrevin 2
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Syntaxin-binding protein 1

MacromoleculeName: Syntaxin-binding protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 68.714883 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSPGISGGGG GIHMAPIGLK AVVGEKIMHD VIKKVKKKGE WKVLVVDQLS MRMLSSCCKM TDIMTEGITI VEDINKRREP LPSLEAVYL ITPSEKSVHS LISDFKDPPT AKYRAAHVFF TDSCPDALFN ELVKSRAAKV IKTLTEINIA FLPYESQVYS L DSADSFQS ...String:
GSPGISGGGG GIHMAPIGLK AVVGEKIMHD VIKKVKKKGE WKVLVVDQLS MRMLSSCCKM TDIMTEGITI VEDINKRREP LPSLEAVYL ITPSEKSVHS LISDFKDPPT AKYRAAHVFF TDSCPDALFN ELVKSRAAKV IKTLTEINIA FLPYESQVYS L DSADSFQS FYSPHKAQMK NPILERLAEQ IATLCATLKE YPAVRYRGEY KDNALLAQLI QDKLDAYKAD DPTMGEGPDK AR SQLLILD RGFDPSSPVL HELTFQAMSY DLLPIENDVY KYETSGIGEA RVKEVLLDED DDLWIALRHK HIAEVSQEVT RSL KDFSSS KRMNTGEKTT MRKLSQMLKK MPQYQKELSK YSTHLHLAED CMKHYQGTVD KLCRVEQDLA MGTDAEGEKI KDPM RAIVP ILLDANVSTY DKIRIILLYI FLKNGITEEN LNKLIQHAQI PPEDSEIITN MAHLGVPIVT DSTLRRRSKP ERKER ISEQ TYQLSRWTPI IKDIMEDTIE DKLDTKHYPY ISTRSSASFS TTAVSARYGH WHKNKAPGEY RSGPRLIIFI LGGVSL NEM RCAYEVTQAN GKWEVLIGST HILTPQKLLD TLKKLNKTDE EISS

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Macromolecule #2: Syntaxin-1A

MacromoleculeName: Syntaxin-1A / type: protein_or_peptide / ID: 2
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
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 29.221541 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSKDRTQELR TAKDSDDDDD VTVTVDRDRF MDEFFEQVEE IRGFIDKIAE NVEEVKRKHS AILASPNPDE KTKEELEELM SDIKKTANK VRSKLKSIEQ SIEQEEGLNR SSADLRIRKT QHSTLSRKFV EVMSEYNATQ SDYRERAKGR IQRQLEITGR T TTSEEAAD ...String:
GSKDRTQELR TAKDSDDDDD VTVTVDRDRF MDEFFEQVEE IRGFIDKIAE NVEEVKRKHS AILASPNPDE KTKEELEELM SDIKKTANK VRSKLKSIEQ SIEQEEGLNR SSADLRIRKT QHSTLSRKFV EVMSEYNATQ SDYRERAKGR IQRQLEITGR T TTSEEAAD MLESGNPAIF ASGIIMDSSI SKQALSEIET RHSEIIKCEN SIRELHDMFM DMAMLVESQG EMIDRIEYNV EH AVDYVER AVSDTKK

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Macromolecule #3: Synaptobrevin-2

MacromoleculeName: Synaptobrevin-2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 6.645386 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSMWNRRLQQ TCAQVDEVVD IMRVNVDKVL ERDQKLSELD DRADALQAGA SQFETSAAK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7.5 mg/mL
BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 7401 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5819182
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: From RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 345463
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION

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