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- PDB-9pdd: 22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydroly... -

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Basic information

Entry
Database: PDB / ID: 9pdd
Title22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydrolyzing, class 29
Components
  • Alpha-soluble NSF attachment protein
  • Unknown SNARE protein
  • 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 / synaptobrevin 2-SNAP-25-syntaxin-1a complex / COPII-mediated vesicle transport / SNARE complex disassembly / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming ...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 / synaptobrevin 2-SNAP-25-syntaxin-1a complex / COPII-mediated vesicle transport / SNARE complex disassembly / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / protein-containing complex disassembly / ATP-dependent protein disaggregase activity / positive regulation of ATP-dependent activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / apical protein localization / syntaxin binding / vesicle-fusing ATPase / syntaxin-1 binding / synaptic vesicle priming / positive regulation of receptor recycling / presynaptic active zone membrane / ionotropic glutamate receptor binding / SNARE binding / synaptic transmission, glutamatergic / PDZ domain binding / neuromuscular junction / intracellular protein transport / brain development / potassium ion transport / neuron differentiation / terminal bouton / positive regulation of protein catabolic process / presynapse / midbody / membrane fusion / postsynapse / protein kinase binding / protein-containing complex binding / glutamatergic synapse / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / plasma membrane / cytosol
Similarity search - Function
NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / 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 ...NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / 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 / PHOSPHATE ION / Vesicle-fusing ATPase / 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: 4.16 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
DepositionJun 30, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 2.0Aug 13, 2025Group: Atomic model / Data collection ...Atomic model / Data collection / Polymer sequence / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / entity / entity_poly / pdbx_validate_torsion
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _em_admin.last_update / _entity.formula_weight / _entity_poly.type
Revision 1.1Aug 13, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / EM metadata / Group: Experimental summary / Other / Structure summary / Data content type: EM metadata / EM metadata / EM metadata / Category: em_admin / entity / entity_poly / Data content type: EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _entity.formula_weight / _entity_poly.type
Revision 2.1Oct 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
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
G: Unknown SNARE protein
I: Alpha-soluble NSF attachment protein
J: Alpha-soluble NSF attachment protein
K: Alpha-soluble NSF attachment protein
L: Alpha-soluble NSF attachment protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)637,63226
Polymers631,73211
Non-polymers5,90115
Water00
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 , 2 types, 10 molecules ABCDEFIJKL

#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
#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

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Protein/peptide , 1 types, 1 molecules G

#2: Protein/peptide Unknown SNARE protein


Mass: 1124.378 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 15 molecules

#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / 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.: 7 / 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.: 2 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25COMPLEX#1-#30MULTIPLE SOURCES
2Subcomplex of alpha-SNAP with poorly defined SNARE bundleCOMPLEX#2-#31RECOMBINANT
3Homohexameric NSFCOMPLEX#11RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Rattus 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 mMmagnesium chlorideMgCl21
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: 33.96 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1.4particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION3.1.4CTF correction
8Coot0.9model fitting
9UCSF ChimeraXmodel fitting
11PHENIX1.21model refinement
12RELION3.1.4initial Euler assignment
13cryoSPARC3.2.0final Euler assignment
14RELION3.1.4classification
15cryoSPARC3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1889717
3D reconstructionResolution: 4.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35805 / Algorithm: EXACT BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MDM

6mdm


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

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