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- PDB-6mdm: The 20S supercomplex engaging the SNAP-25 N-terminus (class 1) -

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Basic information

Entry
Database: PDB / ID: 6mdm
TitleThe 20S supercomplex engaging the SNAP-25 N-terminus (class 1)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / SNARE / NSF / SNAP / ATPase / AAA / disassembly / synapse / membrane fusion / exocytosis
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 / exocytic insertion of neurotransmitter receptor to postsynaptic membrane ...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 / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / myosin head/neck binding / Other interleukin signaling / synaptic vesicle fusion to presynaptic active zone membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / calcium ion-regulated exocytosis of neurotransmitter / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / COPII-mediated vesicle transport / Acetylcholine Neurotransmitter Release Cycle / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Lysosome Vesicle Biogenesis / regulated exocytosis / presynaptic dense core vesicle exocytosis / Dopamine Neurotransmitter Release Cycle / zymogen granule membrane / extrinsic component of presynaptic membrane / ribbon synapse / synaptic vesicle docking / regulation of synaptic vesicle priming / Golgi Associated Vesicle Biogenesis / storage vacuole / regulation of establishment of protein localization / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / response to gravity / positive regulation of calcium ion-dependent exocytosis / vesicle-mediated transport in synapse / protein-containing complex disassembly / vesicle docking / eosinophil degranulation / secretion by cell / SNAP receptor activity / chloride channel inhibitor activity / regulation of exocytosis / SNARE complex / vesicle fusion / neurotransmitter receptor internalization / regulation of vesicle-mediated transport / ATP-dependent protein disaggregase activity / calcium-ion regulated exocytosis / Cargo recognition for clathrin-mediated endocytosis / LGI-ADAM interactions / intra-Golgi vesicle-mediated transport / Clathrin-mediated endocytosis / actomyosin / hormone secretion / positive regulation of intracellular protein transport / Golgi to plasma membrane protein transport / positive regulation of hormone secretion / Golgi stack / neurotransmitter secretion / ATP-dependent protein binding / apical protein localization / neuron projection terminus / positive regulation of ATP-dependent activity / protein localization to membrane / syntaxin binding / vesicle-fusing ATPase / regulation of synaptic vesicle recycling / syntaxin-1 binding / clathrin-coated vesicle / insulin secretion / endosomal transport / SNARE complex assembly / Neutrophil degranulation / positive regulation of neurotransmitter secretion / neurotransmitter transport / synaptic vesicle priming / myosin binding / regulation of synapse assembly / regulation of neuron projection development / positive regulation of receptor recycling / exocytosis / modulation of excitatory postsynaptic potential / associative learning / synaptic vesicle exocytosis / positive regulation of exocytosis / postsynaptic cytosol / protein sumoylation / positive regulation of excitatory postsynaptic potential / synaptic vesicle endocytosis / voltage-gated potassium channel activity / long-term memory / calcium channel inhibitor activity / response to glucose / axonal growth cone
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 / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / Synaptobrevin / 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / 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: 4.4 Å
AuthorsWhite, K.I. / Zhao, M. / Brunger, A.T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R37MH63105 United States
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.
History
DepositionSep 4, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 26, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 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
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

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Structure visualization

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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
Polymers650,38011
Non-polymers5,41911
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 11 molecules ABCDEFHIJKL

#1: Protein
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 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 Synaptosomal-associated protein 25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 23512.387 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
#3: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 29634.129 Da / Num. of mol.: 1
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
#4: Protein Vesicle-associated membrane protein 2 / VAMP-2 / Synaptobrevin-2


Mass: 12981.304 Da / Num. of mol.: 1
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
#5: Protein Alpha-soluble NSF attachment protein / SNAP-alpha / N-ethylmaleimide-sensitive factor attachment protein alpha


Mass: 35598.223 Da / Num. of mol.: 2
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

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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 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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
IDNameTypeEntity IDParent-IDSource
120S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)COMPLEX#1-#50MULTIPLE SOURCES
2N-ethylmaleimide sensitive factorCOMPLEX#11RECOMBINANT
3Synaptosomal-associated protein 25COMPLEX#21RECOMBINANT
4Syntaxin-1ACOMPLEX#31RECOMBINANT
5Vesicle-associated membrane protein 2COMPLEX#41RECOMBINANT
6Alpha-soluble NSF attachment proteinCOMPLEX#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.628533 MDaNO
210.496812 MDaNO
310.023625 MDaNO
410.030726 MDaNO
510.010870 MDaNO
610.033250 MDaNO
710.033250 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33Rattus norvegicus (Norway rat)10116
44Rattus norvegicus (Norway rat)10116
55Rattus norvegicus (Norway rat)10116
66Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
55Escherichia coli (E. coli)562
66Escherichia coli (E. coli)562
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 supportDetails: Not available
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blot for 3.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / 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) / Num. of grids imaged: 2 / Num. 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 selectionNum. of particles selected: 475680
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55150 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3J96

3j96


Accession code: 3J96 / Source name: PDB / Type: experimental model

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