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- EMDB-70608: 22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), 4:2:2 a... -

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Basic information

Entry
Database: EMDB / ID: EMD-70608
Title22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), 4:2:2 alphaSNAP-syntaxin-1a-SNAP-25 subcomplex local refinement, hydrolyzing, class 23
Map dataSharpened map from cryoSPARC local_refine_new, B = 209.8
Sample
  • Complex: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25
    • Complex: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25, 4:2:2
      • Complex: 2:2 binary complex of SNAP-25 and syntaxin-1a
        • Protein or peptide: Syntaxin-1A
        • Protein or peptide: Synaptosomal-associated protein 25
      • Protein or peptide: Alpha-soluble NSF attachment protein
    • Complex: Homohexameric NSF
  • Ligand: water
KeywordsATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion / HYDROLASE
Function / homology
Function and homology information


Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / soluble NSF attachment protein activity / BLOC-1 complex / myosin head/neck binding / synaptic vesicle fusion to presynaptic active zone membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex ...Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / soluble NSF attachment protein activity / BLOC-1 complex / myosin head/neck binding / synaptic vesicle fusion to presynaptic active zone membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / extrinsic component of presynaptic membrane / calcium ion-regulated exocytosis of neurotransmitter / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / COPII-mediated vesicle transport / GABA synthesis, release, reuptake and degradation / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / SNARE complex disassembly / regulated exocytosis / Dopamine Neurotransmitter Release Cycle / synaptic vesicle docking / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / protein-containing complex disassembly / regulation of establishment of protein localization / positive regulation of calcium ion-dependent exocytosis / ribbon synapse / vesicle docking / regulation of exocytosis / secretion by cell / chloride channel inhibitor activity / SNAP receptor activity / SNARE complex / calcium-ion regulated exocytosis / vesicle fusion / actomyosin / hormone secretion / LGI-ADAM interactions / positive regulation of ATP-dependent activity / positive regulation of hormone secretion / ATP-dependent protein binding / neurotransmitter secretion / apical protein localization / protein localization to membrane / syntaxin binding / insulin secretion / syntaxin-1 binding / endosomal transport / Neutrophil degranulation / positive regulation of neurotransmitter secretion / SNARE complex assembly / neurotransmitter transport / regulation of synapse assembly / response to gravity / synaptic vesicle priming / myosin binding / regulation of neuron projection development / exocytosis / associative learning / modulation of excitatory postsynaptic potential / positive regulation of exocytosis / synaptic vesicle exocytosis / protein sumoylation / synaptic vesicle endocytosis / voltage-gated potassium channel activity / positive regulation of excitatory postsynaptic potential / long-term memory / axonal growth cone / calcium channel inhibitor activity / presynaptic active zone membrane / voltage-gated potassium channel complex / somatodendritic compartment / photoreceptor inner segment / endomembrane system / acrosomal vesicle / axonogenesis / secretory granule / SNARE binding / filopodium / synaptic transmission, glutamatergic / intracellular protein transport / neuromuscular junction / locomotory behavior / trans-Golgi network / brain development / postsynaptic density membrane / positive regulation of insulin secretion / kinase binding / Schaffer collateral - CA1 synapse / long-term synaptic potentiation / neuron differentiation / terminal bouton / calcium-dependent protein binding
Similarity search - Function
NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Syntaxin ...NSF attachment protein / Soluble NSF attachment protein, SNAP / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.14 Å
AuthorsWhite KI / Brunger AT
Funding support United States, 3 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Helen Hay Whitney Foundation United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH063105 United States
CitationJournal: bioRxiv / Year: 2025
Title: Structural remodeling of target-SNARE protein complexes by NSF enables synaptic transmission.
Authors: K Ian White / Yousuf A Khan / Kangqiang Qiu / Ashwin Balaji / Sergio Couoh-Cardel / Luis Esquivies / Richard A Pfuetzner / Jiajie Diao / Axel T Brunger /
Abstract: Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and ...Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.
History
DepositionMay 13, 2025-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70608.png

Downloads & links

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Map

FileDownload / File: emd_70608.map.gz / Format: CCP4 / Size: 96.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from cryoSPARC local_refine_new, B = 209.8
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 294 pix.
= 322.224 Å		1.1 Å/pix.
x 294 pix.
= 322.224 Å		1.1 Å/pix.
x 294 pix.
= 322.224 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.096 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.6721399 - 2.0150626
Average (Standard dev.)0.0018131282 (±0.039176106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions294294294
Spacing294294294
CellA=B=C: 322.224 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map from cryoSPARC local refine new

Fileemd_70608_additional_1.map
AnnotationUnsharpened map from cryoSPARC local_refine_new
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B from cryoSPARC local refine new

Fileemd_70608_half_map_1.map
AnnotationHalf map B from cryoSPARC local_refine_new
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A from cryoSPARC local refine new

Fileemd_70608_half_map_2.map
AnnotationHalf map A from cryoSPARC local_refine_new
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binar...

EntireName: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25
Components
  • Complex: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25
    • Complex: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25, 4:2:2
      • Complex: 2:2 binary complex of SNAP-25 and syntaxin-1a
        • Protein or peptide: Syntaxin-1A
        • Protein or peptide: Synaptosomal-associated protein 25
      • Protein or peptide: Alpha-soluble NSF attachment protein
    • Complex: Homohexameric NSF
  • Ligand: water

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Supramolecule #1: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binar...

SupramoleculeName: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25, 4:2:2

SupramoleculeName: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25, 4:2:2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: Homohexameric NSF

SupramoleculeName: Homohexameric NSF / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #4: 2:2 binary complex of SNAP-25 and syntaxin-1a

SupramoleculeName: 2:2 binary complex of SNAP-25 and syntaxin-1a / type: complex / ID: 4 / Parent: 2 / Macromolecule list: #1-#2
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Syntaxin-1A

MacromoleculeName: Syntaxin-1A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 31.045934 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKDRTQELRT AKDSDDDDDV TVTVDRDRFM DEFFEQVEEI RGFIDKIAEN VEEVKRKHSA ILASPNPDEK TKEELEELMS DIKKTANKV RSKLKSIEQS IEQEEGLNRS SADLRIRKTQ HSTLSRKFVE VMSEYNATQS DYRERCKGRI QRQLEITGRT T TSEELEDM ...String:
MKDRTQELRT AKDSDDDDDV TVTVDRDRFM DEFFEQVEEI RGFIDKIAEN VEEVKRKHSA ILASPNPDEK TKEELEELMS DIKKTANKV RSKLKSIEQS IEQEEGLNRS SADLRIRKTQ HSTLSRKFVE VMSEYNATQS DYRERCKGRI QRQLEITGRT T TSEELEDM LESGNPAIFA SGIIMDSSIS KQALSEIETR HSEIIKLENS IRELHDMFMD MAMLVESQGE MIDRIEYNVE HA VDYVERA VSDTKKAVKY QSKARRKKIM

UniProtKB: Syntaxin-1A

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Macromolecule #2: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 25.036746 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQDPNSMAED ADMRNELEEM QRRADQLADE SLESTRRMLQ LVEESKDAGI RTLVMLDEQG EQLERIEEGM DQINKDMKE AEKNLTDLGK FAGLAVAPAN KLKSSDAYKK AWGNNQDGVV ASQPARVVDE REQMAISGGF IRRVTNDARE N EMDENLEQ ...String:
MGSSHHHHHH SQDPNSMAED ADMRNELEEM QRRADQLADE SLESTRRMLQ LVEESKDAGI RTLVMLDEQG EQLERIEEGM DQINKDMKE AEKNLTDLGK FAGLAVAPAN KLKSSDAYKK AWGNNQDGVV ASQPARVVDE REQMAISGGF IRRVTNDARE N EMDENLEQ VSGIIGNLRH MALDMGNEID TQNRQIDRIM EKADSNKTRI DEANQRATKM LGSG

UniProtKB: Synaptosomal-associated protein 25

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Macromolecule #3: Alpha-soluble NSF attachment protein

MacromoleculeName: Alpha-soluble NSF attachment protein / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 33.290715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GMDTSGKQAE AMALLAEAER KVKNSQSFFS GLFGGSSKIE EACEIYARAA NMFKMAKNWS AAGNAFCQAA QLHLQLQSKH DAATCFVDA GNAFKKADPQ EAINCLMRAI EIYTDMGRFT IAAKHHISIA EIYETELVDV EKAIAHYEQS ADYYKGEESN S SANKCLLK ...String:
GMDTSGKQAE AMALLAEAER KVKNSQSFFS GLFGGSSKIE EACEIYARAA NMFKMAKNWS AAGNAFCQAA QLHLQLQSKH DAATCFVDA GNAFKKADPQ EAINCLMRAI EIYTDMGRFT IAAKHHISIA EIYETELVDV EKAIAHYEQS ADYYKGEESN S SANKCLLK VAGYAAQLEQ YQKAIDIYEQ VGTSAMDSPL LKYSAKDYFF KAALCHFCID MLNAKLAVQK YEELFPAFSD SR ECKLMKK LLEAHEEQNV DSYTESVKEY DSISRLDQWL TTMLLRIKKT IQGDEEDLR

UniProtKB: Alpha-soluble NSF attachment protein

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 63 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration15 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris-HCl
150.0 mMsodium chlorideNaCl
1.0 mMmagnesium chlorideMgCl2
1.0 mMadenosine triphosphate
1.0 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER
Details: 15 mA in PELCO easiGlow Glow Discharge Cleaning System
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
SoftwareName: SerialEM
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Average electron dose: 33.96 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Average electron dose: 36.96 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
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

Image recording ID1
Particle selectionNumber selected: 1889717
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 3.1.4)) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: local_refine_new / Number images used: 325236
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.4) / Software - details: InitialModel
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: local_refine_new
Final 3D classificationSoftware - Name: RELION (ver. 3.1.4) / Software - details: Class3D
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6mdm

source_name: PDB, initial_model_type: experimental model

1jth

source_name: PDB, initial_model_type: experimental model
SoftwareName: UCSF ChimeraX (ver. 1.9)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9om6:
22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), 4:2:2 alphaSNAP-syntaxin-1a-SNAP-25 subcomplex local refinement, hydrolyzing, class 23

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