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Basic information

Entry
Database: PDB / ID: 3j99
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy (State IIIb)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / vesicle trafficking
Function / homology
Function and homology information


soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / trans-Golgi Network Vesicle Budding / BLOC-1 complex / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding ...soluble NSF attachment protein activity / Intra-Golgi traffic / Retrograde transport at the Trans-Golgi-Network / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / trans-Golgi Network Vesicle Budding / BLOC-1 complex / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / extrinsic component of presynaptic membrane / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / Lysosome Vesicle Biogenesis / positive regulation of norepinephrine secretion / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / positive regulation of catecholamine secretion / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / protein-containing complex disassembly / Dopamine Neurotransmitter Release Cycle / Golgi Associated Vesicle Biogenesis / zymogen granule membrane / regulated exocytosis / ribbon synapse / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / regulation of synaptic vesicle priming / storage vacuole / regulation of establishment of protein localization / response to gravity / vesicle-mediated transport in synapse / positive regulation of calcium ion-dependent exocytosis / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / calcium ion-regulated exocytosis of neurotransmitter / vesicle fusion / eosinophil degranulation / vesicle docking / chloride channel inhibitor activity / secretion by cell / ATP-dependent protein disaggregase activity / SNARE complex / SNAP receptor activity / Cargo recognition for clathrin-mediated endocytosis / regulation of exocytosis / regulation of vesicle-mediated transport / Clathrin-mediated endocytosis / LGI-ADAM interactions / calcium-ion regulated exocytosis / hormone secretion / intra-Golgi vesicle-mediated transport / actomyosin / positive regulation of intracellular protein transport / Golgi to plasma membrane protein transport / apical protein localization / Golgi stack / neurotransmitter secretion / positive regulation of hormone secretion / neurotransmitter receptor internalization / protein localization to membrane / ATP-dependent protein binding / neuron projection terminus / positive regulation of ATP-dependent activity / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / insulin secretion / neurotransmitter transport / syntaxin binding / syntaxin-1 binding / clathrin-coated vesicle / SNARE complex assembly / positive regulation of neurotransmitter secretion / Neutrophil degranulation / endosomal transport / synaptic vesicle priming / regulation of synapse assembly / postsynaptic cytosol / myosin binding / positive regulation of receptor recycling / regulation of neuron projection development / positive regulation of exocytosis / modulation of excitatory postsynaptic potential / associative learning / exocytosis / synaptic vesicle exocytosis / protein sumoylation / positive regulation of excitatory postsynaptic potential / voltage-gated potassium channel activity / synaptic vesicle endocytosis / endomembrane system / calcium channel inhibitor activity / long-term memory / response to glucose
Similarity search - Function
NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Synaptobrevin/Vesicle-associated membrane protein / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Synaptobrevin signature. ...NSF attachment protein / Soluble NSF attachment protein, SNAP / : / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Synaptobrevin/Vesicle-associated membrane protein / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Synaptobrevin-like / Syntaxin/epimorphin, conserved site / Syntaxin / Synaptobrevin / Syntaxin / epimorphin family signature. / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / SNARE / 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 / 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
Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 2
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsZhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T.
CitationJournal: Nature / Year: 2015
Title: Mechanistic insights into the recycling machine of the SNARE complex.
Authors: Minglei Zhao / Shenping Wu / Qiangjun Zhou / Sandro Vivona / Daniel J Cipriano / Yifan Cheng / Axel T Brunger /
Abstract: Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N- ...Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
History
DepositionDec 5, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 28, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 11, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / refine / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _refine.ls_d_res_high / _refine.ls_d_res_low / _struct_ref_seq_dif.details

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Structure visualization

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  • Deposited structure unit
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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: Alpha-soluble NSF attachment protein
I: Alpha-soluble NSF attachment protein
J: Alpha-soluble NSF attachment protein
G: Alpha-soluble NSF attachment protein
K: Vesicle-associated membrane protein 2
L: Syntaxin-1A
M: Synaptosomal-associated protein 25


Theoretical massNumber of molelcules
Total (without water)667,28213
Polymers667,28213
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Vesicle-fusing ATPase / N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein ...N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein NSF / N-ethylmaleimide sensitive factor


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 / alpha-SNAP


Mass: 33377.793 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 Vesicle-associated membrane protein 2 / VAMP-2 / Synaptobrevin-2


Mass: 7231.061 Da / Num. of mol.: 1 / Fragment: UNP residues 28-89
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli (E. coli) / References: UniProt: P63045
#4: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 7837.957 Da / Num. of mol.: 1 / Fragment: UNP residues 191-256
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
#5: Protein Synaptosomal-associated protein 25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 21256.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881*PLUS

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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
IDNameTypeDetailsParent-ID
120S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)RIBOSOMEOne hexamer of NSF + four alpha-SNAP molecules + one SNARE complex0
2N-ethylmaleimide sensitive factor1
3alpha Soluble NSF Attachment Protein1
4Syntaxin-1A1
5Synaptobrevin-21
6Synaptosomal-associated protein 251
Molecular weightValue: 0.66 MDa / Experimental value: NO
Buffer solutionName: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
pH: 8
Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Holey carbon on top of 400 mesh copper grid
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 77 K / Humidity: 100 %
Details: Blot for 3.5 seconds before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 3.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 28, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: unspecified
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3RELION3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14991 / Nominal pixel size: 2.4312 Å / Actual pixel size: 2.4312 Å
Details: (Single particle details: 3D classification, refinement, and reconstruction were performed using RELION) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: R-factor
Details: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. ...Details: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
11NSF

1nsf

A1NSF1
21QCS

1qcs

A1QCS2
31N7S

1n7s

A1N7S3
41N7S

1n7s

B1N7S3
51N7S

1n7s

C1N7S3
61N7S

1n7s

D1N7S3
RefinementResolution: 8.2→8.2 Å / SU ML: 1.57 / σ(F): 0.79 / Phase error: 39.71 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3037 5618 4.94 %
Rwork0.3059 108021 -
obs0.3058 113639 99.42 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 50 Å2 / Biso mean: 50 Å2 / Biso min: 50 Å2
Refinement stepCycle: LAST / Resolution: 8.201→311.194 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms40976 0 0 0 40976
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00942100
ELECTRON MICROSCOPYf_angle_d1.5756847
ELECTRON MICROSCOPYf_chiral_restr0.0746546
ELECTRON MICROSCOPYf_plane_restr0.0067356
ELECTRON MICROSCOPYf_dihedral_angle_d15.88115533
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
8.2007-8.29390.48222040.443235403744100
8.2939-8.39150.45441680.430836603828100
8.3915-8.49380.45521560.424634773633100
8.4938-8.60130.48282280.425137844012100
8.6013-8.71450.48321560.402933993555100
8.7145-8.83390.3741920.408139064098100
8.8339-8.96010.47911680.411934233591100
8.9601-9.09390.41951920.384736163808100
9.0939-9.2360.42022160.377435853801100
9.236-9.38740.39192280.356737203948100
9.3874-9.54930.38131680.34537153883100
9.5493-9.72290.36242160.325336543870100
9.7229-9.910.38171920.333233393531100
9.91-10.11230.35291580.32643717387599
10.1123-10.33210.32231910.31983599379099
10.3321-10.57250.31471990.3213522372199
10.5725-10.83690.30642040.29473621382599
10.8369-11.12990.27991780.28083573375199
11.1299-11.45750.32681910.28373645383699
11.4575-11.82730.34362020.29113499370199
11.8273-12.250.36831810.30293629381099
12.25-12.74050.33731900.3163523371399
12.7405-13.32030.38231900.34073693388399
13.3203-14.02260.3371650.343566373199
14.0226-14.90120.34632130.31673576378999
14.9012-16.05160.34441440.32533638378299
16.0516-17.66690.33591800.34413573375399
17.6669-20.22290.31321900.34433638382899
20.2229-25.47690.39671560.32673586374299
25.4769-311.41810.15652020.18933605380799

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