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- EMDB-71530: 22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydroly... -

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Basic information

Entry
Database: EMDB / ID: EMD-71530
Title22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydrolyzing, class 22
Map dataSharpened map from CryoSPARC NU-Refine, B = 119.7
Sample
  • Complex: The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25
    • Complex: Unknown SNARE protein
      • Protein or peptide: Vesicle-fusing ATPase
    • Complex: Homohexameric NSF
  • Protein or peptide: unknown sequence
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion / HYDROLASE
Function / homology
Function and homology information


SNARE complex disassembly / ATP-dependent protein disaggregase activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / vesicle-fusing ATPase / syntaxin-1 binding / positive regulation of receptor recycling / ionotropic glutamate receptor binding / SNARE binding ...SNARE complex disassembly / ATP-dependent protein disaggregase activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / vesicle-fusing ATPase / syntaxin-1 binding / positive regulation of receptor recycling / ionotropic glutamate receptor binding / SNARE binding / PDZ domain binding / intracellular protein transport / potassium ion transport / positive regulation of protein catabolic process / midbody / protein kinase binding / protein-containing complex binding / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / plasma membrane / cytosol
Similarity search - Function
: / 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 ...: / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Vesicle-fusing ATPase
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.83 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: 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
DepositionJun 30, 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_71530.png

Downloads & links

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Map

FileDownload / File: emd_71530.map.gz / Format: CCP4 / Size: 96.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from CryoSPARC NU-Refine, B = 119.7
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-1.3761594 - 2.3315396
Average (Standard dev.)0.00063097756 (±0.06781388)
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 NU-Refine

Fileemd_71530_additional_1.map
AnnotationUnsharpened map from CryoSPARC NU-Refine
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A from CryoSPARC NU-Refine

Fileemd_71530_half_map_1.map
AnnotationHalf map A from CryoSPARC NU-Refine
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B from CryoSPARC NU-Refine

Fileemd_71530_half_map_2.map
AnnotationHalf map B from CryoSPARC NU-Refine
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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: Unknown SNARE protein
      • Protein or peptide: Vesicle-fusing ATPase
    • Complex: Homohexameric NSF
  • Protein or peptide: unknown sequence
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION
  • 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

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Supramolecule #2: Unknown SNARE protein

SupramoleculeName: Unknown SNARE protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
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: Cricetulus griseus (Chinese hamster)

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Macromolecule #1: Vesicle-fusing ATPase

MacromoleculeName: Vesicle-fusing ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Cricetulus griseus (Chinese hamster)
Molecular weightTheoretical: 82.90743 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAHMAGRSMQ AARCPTDELS LSNCAVVSEK DYQSGQHVIV RTSPNHKYIF TLRTHPSVVP GSVAFSLPQR KWAGLSIGQE IEVALYSFD KAKQCIGTMT IEIDFLQKKN IDSNPYDTDK MAAEFIQQFN NQAFSVGQQL VFSFNDKLFG LLVKDIEAMD P SILKGEPA ...String:
GAHMAGRSMQ AARCPTDELS LSNCAVVSEK DYQSGQHVIV RTSPNHKYIF TLRTHPSVVP GSVAFSLPQR KWAGLSIGQE IEVALYSFD KAKQCIGTMT IEIDFLQKKN IDSNPYDTDK MAAEFIQQFN NQAFSVGQQL VFSFNDKLFG LLVKDIEAMD P SILKGEPA SGKRQKIEVG LVVGNSQVAF EKAENSSLNL IGKAKTKENR QSIINPDWNF EKMGIGGLDK EFSDIFRRAF AS RVFPPEI VEQMGCKHVK GILLYGPPGC GKTLLARQIG KMLNAREPKV VNGPEILNKY VGESEANIRK LFADAEEEQR RLG ANSGLH IIIFDEIDAI CKQRGSMAGS TGVHDTVVNQ LLSKIDGVEQ LNNILVIGMT NRPDLIDEAL LRPGRLEVKM EIGL PDEKG RLQILHIHTA RMRGHQLLSA DVDIKELAVE TKNFSGAELE GLVRAAQSTA MNRHIKASTK VEVDMEKAES LQVTR GDFL ASLENDIKPA FGTNQEDYAS YIMNGIIKWG DPVTRVLDDG ELLVQQTKNS DRTPLVSVLL EGPPHSGKTA LAAKIA EES NFPFIKICSP DKMIGFSETA KCQAMKKIFD DAYKSQLSCV VVDDIERLLD YVPIGPRFSN LVLQALLVLL KKAPPQG RK LLIIGTTSRK DVLQEMEMLN AFSTTIHVPN IATGEQLLEA LELLGNFKDK ERTTIAQQVK GKKVWIGIKK LLMLIEMS L QMDPEYRVRK FLALLREEGA SPLDFD

UniProtKB: Vesicle-fusing ATPase

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Macromolecule #2: unknown sequence

MacromoleculeName: unknown sequence / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cricetulus griseus (Chinese hamster)
Molecular weightTheoretical: 1.209482 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 51 / 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 33.96 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

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: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 55876
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mdm


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9pdb:
22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydrolyzing, class 22

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