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- PDB-6mdn: The 20S supercomplex engaging the SNAP-25 N-terminus (class 2) -

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Basic information

Entry
Database: PDB / ID: 6mdn
TitleThe 20S supercomplex engaging the SNAP-25 N-terminus (class 2)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25SNAP25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2Vesicle-associated membrane protein
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / SNARE / NSF / SNAP / ATPase / AAA / disassembly / synapse / membrane fusion / exocytosis
Function / homologyAAA+ lid domain / SNARE / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2 / Syntaxin 1A ...AAA+ lid domain / SNARE / Cell division protein 48 (CDC48), N-terminal domain / Synaptobrevin / SNAP-25 family / Syntaxin / ATPase family associated with various cellular activities (AAA) / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2 / Syntaxin 1A / P-loop containing nucleoside triphosphate hydrolase / Synaptobrevin/Vesicle-associated membrane protein / Tetratricopeptide-like helical domain superfamily / Aspartate decarboxylase-like domain superfamily / SNARE domain / Syntaxin/epimorphin, conserved site / Syntaxin, N-terminal domain / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / Synaptobrevin / SNAP-25 / NSF attachment protein / Target SNARE coiled-coil homology domain / Cell division protein 48 (CDC48), domain 2 / Synaptosomal-associated protein 25 / Synaptobrevin signature. / Dopamine Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Clathrin-mediated endocytosis / AAA-protein family signature. / Retrograde transport at the Trans-Golgi-Network / Intra-Golgi traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / LGI-ADAM interactions / Other interleukin signaling / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / Clathrin derived vesicle budding / Acetylcholine Neurotransmitter Release Cycle / Cargo recognition for clathrin-mediated endocytosis / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Syntaxin / epimorphin family signature. / v-SNARE coiled-coil homology domain profile. / Serotonin Neurotransmitter Release Cycle / t-SNARE coiled-coil homology domain profile. / COPII-mediated vesicle transport / soluble NSF attachment protein activity / SNARE complex disassembly / protein-containing complex disassembly / regulation of delayed rectifier potassium channel activity / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / myosin head/neck binding / regulation of synaptic vesicle priming / zymogen granule membrane / presynaptic active zone membrane / synaptic vesicle docking / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / neurotransmitter transport / positive regulation of hormone secretion / positive regulation of norepinephrine secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptic vesicle fusion to presynaptic active zone membrane / positive regulation of catecholamine secretion / synaptic vesicle priming / SNARE complex assembly / regulation of synaptic vesicle recycling / intracellular organelle / regulation of synapse assembly / storage vacuole / positive regulation of neurotransmitter secretion / apical protein localization / integral component of synaptic vesicle membrane / Golgi to plasma membrane protein transport / synaptic vesicle exocytosis / calcium ion-regulated exocytosis of neurotransmitter / vesicle-fusing ATPase / response to gravity / protein localization to membrane / positive regulation of calcium ion-dependent exocytosis / chloride channel inhibitor activity / sleep / SNAP receptor activity / regulation of exocytosis / positive regulation of ATPase activity / regulation of neuron projection development / SNARE complex / positive regulation of intracellular protein transport / vacuolar membrane / membrane fusion / syntaxin-1 binding / synaptic transmission, glutamatergic / neuron projection terminus / ATP-dependent protein binding
Function and homology information
Specimen sourceCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.4 Å resolution
AuthorsWhite, K.I. / Zhao, M. / Brunger, A.T.
CitationJournal: Elife / Year: 2018
Title: Structural principles of SNARE complex recognition by the AAA+ protein NSF.
Authors: K Ian White / Minglei Zhao / Ucheor B Choi / Richard A Pfuetzner / Axel T Brunger
Abstract: The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (-ethylmaleimide sensitive ...The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 4, 2018 / Release: Sep 19, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Sep 19, 2018Structure modelrepositoryInitial release
1.1Sep 26, 2018Structure modelData collection / Database referencescitation / citation_author_citation.journal_abbrev / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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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: Synaptosomal-associated protein 25
I: Syntaxin-1A
J: Vesicle-associated membrane protein 2
K: Alpha-soluble NSF attachment protein
L: Alpha-soluble NSF attachment protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)655,79922
Polyers650,38011
Non-polymers5,41911
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein/peptide , 5 types, 11 molecules ABCDEFHIJKL

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


Mass: 85509.227 Da / Num. of mol.: 6 / Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase
#2: Protein/peptide Synaptosomal-associated protein 25 / SNAP25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 23512.387 Da / Num. of mol.: 1 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881
#3: Protein/peptide Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 29634.129 Da / Num. of mol.: 1 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#4: Protein/peptide Vesicle-associated membrane protein 2 / Vesicle-associated membrane protein / VAMP-2 / Synaptobrevin-2


Mass: 12981.304 Da / Num. of mol.: 1 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli (E. coli) / References: UniProt: P63045
#5: Protein/peptide Alpha-soluble NSF attachment protein / SNAP-alpha / N-ethylmaleimide-sensitive factor attachment protein alpha


Mass: 35598.223 Da / Num. of mol.: 2 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P54921

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Non-polymers , 2 types, 11 molecules

#6: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 9 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
120S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)COMPLEX1, 2, 3, 4, 50MULTIPLE SOURCES
2N-ethylmaleimide sensitive factorCOMPLEX11RECOMBINANT
3Synaptosomal-associated protein 25COMPLEX21RECOMBINANT
4Syntaxin-1ACOMPLEX31RECOMBINANT
5Vesicle-associated membrane protein 2COMPLEX41RECOMBINANT
6Alpha-soluble NSF attachment proteinCOMPLEX51RECOMBINANT
Molecular weight
IDValueEntity assembly IDExperimental value
10.628533 MDa1NO
20.496812 MDa2NO
30.023625 MDa3NO
40.030726 MDa4NO
50.010870 MDa5NO
60.033250 MDa6NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
2210029Cricetulus griseus (Chinese hamster)
3310116Rattus norvegicus (Norway rat)
4410116Rattus norvegicus (Norway rat)
5510116Rattus norvegicus (Norway rat)
6610116Rattus norvegicus (Norway rat)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli (E. coli)
33562Escherichia coli (E. coli)
44562Escherichia coli (E. coli)
55562Escherichia coli (E. coli)
66562Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
10.05 MTris HClC4H11NO31
20.150 MSodium chlorideNaCl1
30.001 MAdenosine triphosphateC10H16N5O13P31
40.001 MEthylenediaminetetraacetic acidC10H16N2O81
50.001 MDithiothreitolC4H10O2S21
60.05 % v/vNonidet P-401
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 kelvins / Details: Blot for 3.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 58 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 5418
Image scansMovie frames/image: 40 / Used frames/image: 2-40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION2.1CTF correction
7PHENIXmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11Cootmodel refinement
12RELION2.1initial Euler assignment
13RELION2.1final Euler assignment
14RELION2.1classification
15RELION2.13D reconstruction
CTF correctionDetails: CTF correction was carried out in Relion with reconstruction step.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 475680
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 62723 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 3J96

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