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- EMDB-70548: 21bin20S complex (NSF-alphaSNAP-2:1 syntaxin-1a:SNAP-25), non-hyd... -

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Basic information

Entry
Database: EMDB / ID: EMD-70548
Title21bin20S complex (NSF-alphaSNAP-2:1 syntaxin-1a:SNAP-25), non-hydrolyzing, class 4
Map dataSharpened map from RELION Postprocess, B = -70.7744
Sample
  • Complex: The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binary SNARE complex of syntaxin-1a and SNAP-25
    • Complex: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25
      • Protein or peptide: Synaptosomal-associated protein 25
    • Complex: Homohexameric NSF
      • Protein or peptide: Vesicle-fusing ATPase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: water
KeywordsATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion / HYDROLASE
Function / homology
Function and homology information


BLOC-1 complex / 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 ...BLOC-1 complex / 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 / SNARE complex disassembly / regulation of establishment of protein localization / ribbon synapse / SNARE complex / SNAP receptor activity / ATP-dependent protein disaggregase activity / positive regulation of hormone secretion / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / neurotransmitter secretion / vesicle-fusing ATPase / syntaxin-1 binding / endosomal transport / Neutrophil degranulation / SNARE complex assembly / regulation of synapse assembly / myosin binding / synaptic vesicle priming / regulation of neuron projection development / exocytosis / positive regulation of receptor recycling / associative learning / synaptic vesicle exocytosis / voltage-gated potassium channel activity / long-term memory / axonal growth cone / somatodendritic compartment / voltage-gated potassium channel complex / photoreceptor inner segment / ionotropic glutamate receptor binding / axonogenesis / SNARE binding / filopodium / PDZ domain binding / locomotory behavior / intracellular protein transport / trans-Golgi network / positive regulation of insulin secretion / potassium ion transport / long-term synaptic potentiation / neuron differentiation / terminal bouton / positive regulation of protein catabolic process / calcium-dependent protein binding / synaptic vesicle / lamellipodium / actin cytoskeleton / presynaptic membrane / growth cone / midbody / 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 / cytoplasm / cytosol
Similarity search - Function
: / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / 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 / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / 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
Vesicle-fusing ATPase / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 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: 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 8, 2025-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70548.png

Downloads & links

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Map

FileDownload / File: emd_70548.map.gz / Format: CCP4 / Size: 96.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from RELION Postprocess, B = -70.7744
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: 0.03
Minimum - Maximum-0.10947702 - 0.24600962
Average (Standard dev.)-0.000036058835 (±0.0056064287)
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 RELION Refine3D

Fileemd_70548_additional_1.map
AnnotationUnsharpened map from RELION Refine3D
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from RELION Refine3D

Fileemd_70548_half_map_1.map
AnnotationHalf map 1 from RELION Refine3D
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from RELION Refine3D

Fileemd_70548_half_map_2.map
AnnotationHalf map 2 from RELION Refine3D
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binar...

EntireName: The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binary SNARE complex of syntaxin-1a and SNAP-25
Components
  • Complex: The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binary SNARE complex of syntaxin-1a and SNAP-25
    • Complex: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25
      • Protein or peptide: Synaptosomal-associated protein 25
    • Complex: Homohexameric NSF
      • Protein or peptide: Vesicle-fusing ATPase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: water

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Supramolecule #1: The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binar...

SupramoleculeName: The 21bin20S complex of NSF, alphaSNAP, and the soluble 2:1 binary SNARE complex of syntaxin-1a and SNAP-25
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25

SupramoleculeName: Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
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: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 9.741827 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMAEDADMRN ELEEMQRRAD QLADESLEST RRMLQLVEES KDAGIRTLVM LDEQGEQLER IEEGMDQINK DMKEAEKNLT DLGK

UniProtKB: Synaptosomal-associated protein 25

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 10 / 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: 1 / 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: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 754 / 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 mMEDTA
1.0 mMadenosine triphosphate
1.0 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER
Details: 15 mA in PELCO easiGlow Glow Discharge Cleaning System
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: 31.56 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: 1656248
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 3.1.4)) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
RELION (ver. 3.1.4)Refine3D
RELION (ver. 3.1.4)Postprocess

Number images used: 955526
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.4)
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-9oju:
21bin20S complex (NSF-alphaSNAP-2:1 syntaxin-1a:SNAP-25), non-hydrolyzing, class 4

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