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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
KeywordsMunc18 / syntaxin / synaptobrevin / SNARE / membrane fusion / neurotransmitter release / EXOCYTOSIS
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 / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / extrinsic component of presynaptic membrane / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / positive regulation of norepinephrine secretion / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / positive regulation of catecholamine secretion / Acetylcholine Neurotransmitter Release Cycle / Lysosome Vesicle Biogenesis / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / zymogen granule membrane / regulated exocytosis / negative regulation of synaptic transmission, GABAergic / presynaptic dense core vesicle exocytosis / platelet degranulation / synaptic vesicle docking / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / storage vacuole / response to gravity / vesicle-mediated transport in synapse / positive regulation of calcium ion-dependent exocytosis / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / synaptic vesicle maturation / vesicle fusion / eosinophil degranulation / vesicle docking / neuromuscular synaptic transmission / secretion by cell / SNARE complex / chloride channel inhibitor activity / SNAP receptor activity / presynaptic active zone cytoplasmic component / regulation of exocytosis / positive regulation of mast cell degranulation / regulation of vesicle-mediated transport / Cargo recognition for clathrin-mediated endocytosis / LGI-ADAM interactions / Clathrin-mediated endocytosis / calcium-ion regulated exocytosis / hormone secretion / actomyosin / positive regulation of intracellular protein transport / platelet alpha granule / Golgi to plasma membrane protein transport / neurotransmitter secretion / protein localization to membrane / ATP-dependent protein binding / neuron projection terminus / vesicle docking involved in exocytosis / presynaptic cytosol / regulation of synaptic vesicle recycling / insulin secretion / long-term synaptic depression / syntaxin binding / neurotransmitter transport / syntaxin-1 binding / parallel fiber to Purkinje cell synapse / clathrin-coated vesicle / SNARE complex assembly / positive regulation of neurotransmitter secretion / synaptic vesicle priming / postsynaptic cytosol / myosin binding / modulation of excitatory postsynaptic potential / exocytosis / synaptic vesicle exocytosis / positive regulation of exocytosis / phospholipase binding / protein sumoylation / positive regulation of excitatory postsynaptic potential / synaptic vesicle endocytosis / negative regulation of protein-containing complex assembly / calcium channel inhibitor activity / endomembrane system / response to glucose / phagocytic vesicle / presynaptic active zone membrane / vesicle-mediated transport / acrosomal vesicle / SNARE binding / secretory granule / protein localization to plasma membrane / establishment of localization in cell
Similarity search - Function
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 ...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/epimorphin, conserved site / Syntaxin / 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing 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
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 180 pix.
= 194.4 Å		1.08 Å/pix.
x 180 pix.
= 194.4 Å		1.08 Å/pix.
x 180 pix.
= 194.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
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 / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)

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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

UniProtKB: Syntaxin-binding protein 1

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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

UniProtKB: Syntaxin-1A

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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

UniProtKB: Vesicle-associated membrane protein 2

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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
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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