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- PDB-9oj2: sx20S complex (NSF-alphaSNAP-syntaxin-1a), non-hydrolyzing, class 1 -

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Basic information

Entry
Database: PDB / ID: 9oj2
Titlesx20S complex (NSF-alphaSNAP-syntaxin-1a), non-hydrolyzing, class 1
Components
  • Alpha-soluble NSF attachment protein
  • Syntaxin-1A
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / ATPase / ATPases Associated with diverse cellular Activities / AAA+ / 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 / lamellar body / myosin head/neck binding / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I 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 / lamellar body / myosin head/neck binding / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I 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 / protein-containing complex disassembly / positive regulation of catecholamine secretion / positive regulation of norepinephrine secretion / Dopamine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / hormone secretion / regulation of synaptic vesicle priming / Golgi Associated Vesicle Biogenesis / : / regulated exocytosis / secretion by cell / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / calcium-ion regulated exocytosis / SNARE complex disassembly / positive regulation of calcium ion-dependent exocytosis / positive regulation of neurotransmitter secretion / : / chloride channel inhibitor activity / regulation of exocytosis / protein carrier activity / SNARE complex / SNAP receptor activity / vesicle fusion / intra-Golgi vesicle-mediated transport / actomyosin / LGI-ADAM interactions / Golgi to plasma membrane protein transport / positive regulation of ATP-dependent activity / ATP-dependent protein disaggregase activity / Golgi stack / ATP-dependent protein binding / cytoplasmic side of membrane / insulin secretion / apical protein localization / syntaxin binding / protein localization to membrane / vesicle-fusing ATPase / exocytosis / myosin binding / SNARE complex assembly / synaptic vesicle priming / response to gravity / synaptic vesicle exocytosis / positive regulation of receptor recycling / neurotransmitter transport / positive regulation of exocytosis / response to hyperoxia / regulation of dopamine secretion / modulation of excitatory postsynaptic potential / protein sumoylation / synaptic vesicle endocytosis / calcium channel inhibitor activity / presynaptic active zone membrane / secretory granule / ionotropic glutamate receptor binding / acrosomal vesicle / positive regulation of excitatory postsynaptic potential / brain development / SNARE binding / synaptic membrane / neuromuscular junction / synaptic transmission, glutamatergic / PDZ domain binding / intracellular protein transport / postsynaptic density membrane / Schaffer collateral - CA1 synapse / kinase binding / calcium-dependent protein binding / terminal bouton / positive regulation of protein catabolic process / synaptic vesicle membrane / neuron differentiation / presynapse / synaptic vesicle / nuclear membrane / midbody / cytoplasmic vesicle / presynaptic membrane / membrane fusion / perikaryon / transmembrane transporter binding / postsynapse / protein-macromolecule adaptor activity / postsynaptic membrane
Similarity search - Function
NSF attachment protein / Soluble NSF attachment protein, SNAP / Vesicle-fusing ATPase / : / NSF, AAA+ ATPase lid domain / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / Syntaxin/epimorphin, conserved site ...NSF attachment protein / Soluble NSF attachment protein, SNAP / Vesicle-fusing ATPase / : / NSF, AAA+ ATPase lid domain / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / SNARE domain / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), N-terminal domain / 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 / PHOSPHATE ION / Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 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
CitationJournal: 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.
History
DepositionMay 6, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 23, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 23, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)754,11227
Polymers748,01114
Non-polymers6,10113
Water1,67593
1
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)503,54619
Polymers497,4456
Non-polymers6,10113
Water1086
TypeNameSymmetry operationNumber
identity operation1_5551
2
K: Alpha-soluble NSF attachment protein
M: Alpha-soluble NSF attachment protein
H: Syntaxin-1A
G: Syntaxin-1A
I: Syntaxin-1A
J: Syntaxin-1A
N: Alpha-soluble NSF attachment protein
L: Alpha-soluble NSF attachment protein


Theoretical massNumber of molelcules
Total (without water)250,5668
Polymers250,5668
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 14 molecules ABCDEFKMNLHGIJ

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


Mass: 29350.803 Da / Num. of mol.: 4 / Fragment: residues 1-253
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

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Non-polymers , 4 types, 106 molecules

#4: 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
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 93 / 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 sx20S complex of NSF, alphaSNAP, and soluble syntaxin-1aCOMPLEX#1-#30MULTIPLE SOURCES
2Homohexameric NSFCOMPLEX#11RECOMBINANT
3Subcomplex of alphaSNAP-syntaxin-1aCOMPLEX#2-#31RECOMBINANT
4Homotetrameric syntaxin-1aCOMPLEX#33RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
24
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33Rattus norvegicus (Norway rat)10116
44Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA with PELCO easiGlow Glow Discharge Cleaning System
Grid 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: 27.608 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION3.1.4CTF correction
8Coot0.9model fitting
9UCSF ChimeraXmodel fitting
11RELION3.1.4initial Euler assignment
12cryoSPARC3.2.0final Euler assignmentNU-Refine
13RELION3.1.4classification
14cryoSPARC3.2.03D reconstruction
15PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 975203
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53244 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / 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
21N7S

1n7s

11N7S2PDBexperimental model

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