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- PDB-3j96: Structure of 20S supercomplex determined by single particle cryoe... -

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Basic information

Entry
Database: PDB / ID: 3j96
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy (State I)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25SNAP25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2Vesicle-associated membrane protein
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / vesicle trafficking
Function / homology
Function and homology information


soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / anchored component of presynaptic membrane / zymogen granule membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / eosinophil degranulation / hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex ...soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / anchored component of presynaptic membrane / zymogen granule membrane / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / eosinophil degranulation / hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex / growth hormone secretion / neurotransmitter transport / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / regulation of synaptic vesicle priming / positive regulation of norepinephrine secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptic vesicle fusion to presynaptic active zone membrane / presynaptic active zone membrane / positive regulation of catecholamine secretion / short-term synaptic potentiation / regulation of establishment of protein localization / synaptic vesicle docking / regulated exocytosis / protein-containing complex disassembly / intracellular organelle / storage vacuole / chloride channel inhibitor activity / calcium ion-regulated exocytosis of neurotransmitter / regulation of synaptic vesicle recycling / neurotransmitter receptor internalization / vesicle docking / apical protein localization / secretion by cell / positive regulation of calcium ion-dependent exocytosis / positive regulation of neurotransmitter secretion / protein localization to membrane / membrane fusion / integral component of synaptic vesicle membrane / Golgi to plasma membrane protein transport / vesicle fusion / vesicle-fusing ATPase / positive regulation of hormone secretion / SNARE complex assembly / regulation of vesicle-mediated transport / SNAP receptor activity / SNARE complex / synaptic vesicle priming / sleep / response to gravity / calcium-ion regulated exocytosis / regulation of exocytosis / positive regulation of ATPase activity / positive regulation of synaptic plasticity / positive regulation of intracellular protein transport / positive regulation of receptor recycling / regulation of synapse assembly / ATP-dependent protein binding / actomyosin / regulation of neuron projection development / syntaxin-1 binding / synaptic vesicle exocytosis / vacuolar membrane / neuron projection terminus / modulation of excitatory postsynaptic potential / myosin binding / positive regulation of exocytosis / neurotransmitter secretion / clathrin-coated vesicle / insulin secretion / syntaxin binding / protein sumoylation / voltage-gated potassium channel complex / ionotropic glutamate receptor binding / exocytosis / synaptic vesicle endocytosis / voltage-gated potassium channel activity / endomembrane system / associative learning / calcium channel inhibitor activity / long-term memory / positive regulation of insulin secretion / somatodendritic compartment / positive regulation of excitatory postsynaptic potential / long-term synaptic potentiation / SNARE binding / axonal growth cone / photoreceptor inner segment / synaptic vesicle membrane / synaptic transmission, glutamatergic / axonogenesis / acrosomal vesicle / locomotory behavior / presynapse / vesicle-mediated transport / filopodium / potassium ion transport / response to glucose / endosomal transport / ATPase activity, coupled / integral component of presynaptic membrane
SNAP-25 family / Synaptobrevin/Vesicle-associated membrane protein / Target SNARE coiled-coil homology domain / NSF attachment protein / SNAP-25 domain / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site ...SNAP-25 family / Synaptobrevin/Vesicle-associated membrane protein / Target SNARE coiled-coil homology domain / NSF attachment protein / SNAP-25 domain / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / CDC48, domain 2 / Syntaxin/epimorphin, conserved site / Aspartate decarboxylase-like domain superfamily / SNARE / Tetratricopeptide-like helical domain superfamily / Syntaxin, N-terminal domain / P-loop containing nucleoside triphosphate hydrolase / AAA ATPase, AAA+ lid domain / AAA+ lid domain / Syntaxin 1 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / v-SNARE, coiled-coil homology domain / Cell division protein 48 (CDC48), domain 2 / Synaptosomal-associated protein 25 / Vesicle-fusing ATPase / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2
Vesicle-fusing ATPase / Syntaxin-1A / Alpha-soluble NSF attachment protein / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 2
Biological speciesCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.6 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.
Validation Report
SummaryFull reportAbout validation report
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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-6206
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Structure viewerMolecule:
MolmilJmol/JSmol

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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: Alpha-soluble NSF attachment protein
H: Alpha-soluble NSF attachment protein
I: Alpha-soluble NSF attachment protein
J: 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
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide
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/peptide
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/peptide Vesicle-associated membrane protein 2 / Vesicle-associated membrane protein / 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/peptide 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/peptide Synaptosomal-associated protein 25 / SNAP25 / 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 25SNAP251
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: 90 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 FIELDBright-field microscopy / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm
Specimen holderModel: 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: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29717 / 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. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
11NSFA
21QCSA
31N7SA
41N7SB
51N7SC
61N7SD
RefinementResolution: 7.601→311.194 Å / SU ML: 1.53 / σ(F): 0.5 / Phase error: 40.44 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3035 7233 5.06 %
Rwork0.2997 135735 -
Obs0.2999 142968 99.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 550 Å2 / Biso mean: 500.074 Å2 / Biso min: 30 Å2
Refinement stepCycle: LAST / Resolution: 7.601→311.194 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms41139 0 0 0 41139
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00942091
f_angle_d1.59656840
f_chiral_restr0.0736546
f_plane_restr0.0067355
f_dihedral_angle_d15.90615528
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
7.6014-7.68780.52832280.499544764704100
7.6878-7.77820.51022520.48249565208100
7.7782-7.87310.50412320.473642214453100
7.8731-7.97280.45142400.464547645004100
7.9728-8.07770.44812520.44442994551100
8.0777-8.18840.43592400.433748585098100
8.1884-8.30530.42482640.420843594623100
8.3053-8.42930.38532310.410645364767100
8.4293-8.56110.43761920.405947024894100
8.5611-8.70140.43432520.400243834635100
8.7014-8.85150.43582520.397345544806100
8.8515-9.01240.39732400.383446154855100
9.0124-9.18580.38882760.354144734749100
9.1858-9.37330.33542280.341945124740100
9.3733-9.57710.35572640.321146054869100
9.5771-9.79990.34522390.307746764915100
9.7999-10.0450.30722400.30424499473999
10.045-10.31660.28812390.30154272451198
10.3166-10.62020.29742270.29064462468998
10.6202-10.9630.28772720.2794568484099
10.963-11.35490.28011980.26664398459698
11.3549-11.80950.28132340.26564508474299
11.8095-12.3470.28032130.27244489470299
12.347-12.9980.29552060.28484607481399
12.998-13.81230.37732860.30264462474899
13.8123-14.87880.32732380.30964464470299
14.8788-16.3760.33122640.30814563482799
16.376-18.74540.35722510.31794437468899
18.7454-23.6160.33762500.32144491474199
23.616-311.4550.16122330.17744526475999

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