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- PDB-9pff: Min22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a H3:SNAP-25 SN1... -

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Basic information

Entry
Database: PDB / ID: 9pff
TitleMin22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a H3:SNAP-25 SN1), non-hydrolyzing, class 27
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25
  • Syntaxin-1A
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / ATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion
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 / Dopamine Neurotransmitter Release Cycle / synaptic vesicle docking / SNARE complex disassembly / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / regulated exocytosis / 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 / SNARE complex / SNAP receptor activity / calcium-ion regulated exocytosis / vesicle fusion / ATP-dependent protein disaggregase activity / actomyosin / hormone secretion / LGI-ADAM interactions / positive regulation of ATP-dependent activity / positive regulation of hormone secretion / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / ATP-dependent protein binding / Golgi stack / neurotransmitter secretion / protein localization to membrane / apical protein localization / syntaxin binding / vesicle-fusing ATPase / insulin secretion / syntaxin-1 binding / endosomal transport / Neutrophil degranulation / SNARE complex assembly / positive regulation of neurotransmitter secretion / neurotransmitter transport / regulation of synapse assembly / myosin binding / synaptic vesicle priming / response to gravity / regulation of neuron projection development / exocytosis / positive regulation of receptor recycling / 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 / somatodendritic compartment / voltage-gated potassium channel complex / photoreceptor inner segment / ionotropic glutamate receptor binding / endomembrane system / acrosomal vesicle / axonogenesis / secretory granule / SNARE binding / filopodium / synaptic transmission, glutamatergic / PDZ domain binding / locomotory behavior / neuromuscular junction / intracellular protein transport / trans-Golgi network / brain development
Similarity search - Function
NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / Syntaxin / Syntaxin N-terminal domain ...NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / 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 / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Tetratricopeptide-like helical domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Cricetulus griseus (Chinese hamster)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsWhite, K.I. / Brunger, A.T.
Funding support United States, 3items
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
Citation
Journal: Nat Commun / 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.
#1: Journal: 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
DepositionJul 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 17, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.title / _citation.year ..._citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Synaptosomal-associated protein 25
H: Syntaxin-1A
I: Synaptosomal-associated protein 25
J: Syntaxin-1A
L: Alpha-soluble NSF attachment protein
M: Alpha-soluble NSF attachment protein
N: Alpha-soluble NSF attachment protein
O: Alpha-soluble NSF attachment protein
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)674,05725
Polymers668,55814
Non-polymers5,49911
Water14,610811
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 14 molecules GIHJLMNOABCDEF

#1: Protein Synaptosomal-associated protein 25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 9741.827 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881
#2: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 9233.696 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#3: Protein
Alpha-soluble NSF attachment protein / SNAP-alpha / N-ethylmaleimide-sensitive factor attachment protein alpha


Mass: 33290.715 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P54921
#4: Protein
Vesicle-fusing ATPase / N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein NSF


Mass: 82907.430 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase

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Non-polymers , 3 types, 822 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 811 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1The min22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of the syntaxin-1a H3 and SNAP-25 SN1 domainsCOMPLEX#1-#40MULTIPLE SOURCES
2Subcomplex of alphaSNAP, syntaxin-1a H3, and SNAP-25 SN1COMPLEX#1-#31RECOMBINANT
3Homohexameric NSFCOMPLEX#41RECOMBINANT
42:2 binary complex of SNAP-25 SN1 and syntaxin-1a H3COMPLEX#2-#32RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Rattus norvegicus (Norway rat)10116
43Cricetulus griseus (Chinese hamster)10029
54Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2150 mMsodium chlorideNaCl1
31 mMEDTA1
41 mMadenosine triphosphate1
51 mMTCEP1
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 33.992 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1.4particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION3.1.4CTF correction
8Coot0.9model fitting
9UCSF ChimeraXmodel fitting
11RELION3.1.4initial Euler assignment
12cryoSPARC3.2.0final Euler assignment
13RELION3.1.4classification
14cryoSPARC3.2.03D reconstructionNU-Refine
15PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1473284
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 341427 / Algorithm: EXACT BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16MDM

6mdm

16MDM1PDBexperimental model
21JTH

1jth

11JTH2PDBexperimental model

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