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- PDB-9ojr: 21bin20S complex (NSF-alphaSNAP-2:1 syntaxin-1a:SNAP-25), non-hyd... -

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Basic information

Entry
Database: PDB / ID: 9ojr
Title21bin20S complex (NSF-alphaSNAP-2:1 syntaxin-1a:SNAP-25), non-hydrolyzing, class 3
Components
  • Synaptosomal-associated protein 25
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / ATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion
Function / homology
Function and homology information


BLOC-1 complex / SNARE complex disassembly / 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 ...BLOC-1 complex / SNARE complex disassembly / 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 / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / regulation of establishment of protein localization / ribbon synapse / SNAP receptor activity / SNARE complex / ATP-dependent protein disaggregase activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / positive regulation of hormone secretion / Golgi stack / neurotransmitter secretion / vesicle-fusing ATPase / syntaxin-1 binding / endosomal transport / Neutrophil degranulation / SNARE complex assembly / synaptic vesicle priming / regulation of synapse assembly / myosin binding / regulation of neuron projection development / positive regulation of receptor recycling / exocytosis / synaptic vesicle exocytosis / associative learning / voltage-gated potassium channel activity / long-term memory / axonal growth cone / photoreceptor inner segment / voltage-gated potassium channel complex / somatodendritic compartment / ionotropic glutamate receptor binding / axonogenesis / SNARE binding / PDZ domain binding / filopodium / intracellular protein transport / locomotory behavior / trans-Golgi network / positive regulation of insulin secretion / potassium ion transport / long-term synaptic potentiation / terminal bouton / neuron differentiation / positive regulation of protein catabolic process / calcium-dependent protein binding / synaptic vesicle / actin cytoskeleton / lamellipodium / presynaptic membrane / midbody / growth cone / cell cortex / vesicle / transmembrane transporter binding / cytoskeleton / neuron projection / endosome / protein domain specific binding / axon / neuronal cell body / synapse / lipid binding / protein kinase binding / protein-containing complex binding / perinuclear region of cytoplasm / glutamatergic synapse / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / 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 ...: / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / 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 / 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 / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Rattus rattus (black rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 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: bioRxiv / Year: 2024
Title: Pre-fusion AAA+ remodeling of target-SNARE protein complexes 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: Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and ...Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. 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 show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or 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 8, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: 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
H: Synaptosomal-associated protein 25
hetero molecules


Theoretical massNumber of molelcules
Total (without water)513,19319
Polymers507,1867
Non-polymers6,00612
Water13,763764
1


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

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Components

#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 Synaptosomal-associated protein 25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 9741.827 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus rattus (black rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881
#3: 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
#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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 764 / Source method: isolated from a natural source / Formula: H2O
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 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binary SNARE complex of syntaxin-1a and SNAP-25COMPLEX#1-#20MULTIPLE SOURCES
2Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25COMPLEX#21RECOMBINANT
3Homohexameric NSFCOMPLEX#11RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Rattus norvegicus (Norway rat)10116
43Cricetulus griseus (Chinese hamster)10029
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli (E. coli)562
43Escherichia 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 supportDetails: 15 mA in 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: 31.56 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
12RELION3.1.4final Euler assignment
13RELION3.1.4classification
14RELION3.1.43D reconstructionRefine3D
15RELION3.1.43D reconstructionPostprocess
22PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1656248
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 637397 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6MDM

6mdm


Accession code: 6MDM / Source name: PDB / Type: experimental model

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