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- PDB-6mdp: The D1 and D2 domain rings of NSF engaging the SNAP-25 N-terminus... -

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Basic information

Entry
Database: PDB / ID: 6mdp
TitleThe D1 and D2 domain rings of NSF engaging the SNAP-25 N-terminus within the 20S supercomplex (focused refinement on D1/D2 rings, class 2)
Components
  • Synaptosomal-associated protein 25SNAP25
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / SNARE / NSF / SNAP / ATPase / AAA / disassembly / synapse / membrane fusion / exocytosis
Function / homologyAAA+ lid domain / P-loop containing nucleoside triphosphate hydrolase / Target SNARE coiled-coil homology domain / SNAP-25 / CDC48, N-terminal subdomain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / CDC48, domain 2 / Aspartate decarboxylase-like domain superfamily / AAA+ ATPase domain ...AAA+ lid domain / P-loop containing nucleoside triphosphate hydrolase / Target SNARE coiled-coil homology domain / SNAP-25 / CDC48, N-terminal subdomain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / CDC48, domain 2 / Aspartate decarboxylase-like domain superfamily / AAA+ ATPase domain / CDC48 domain 2-like superfamily / Vesicle-fusing ATPase / ATPase family associated with various cellular activities (AAA) / SNAP-25 family / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48), domain 2 / AAA-protein family signature. / t-SNARE coiled-coil homology domain profile. / Synaptosomal-associated protein 25 / SNARE complex disassembly / anchored component of presynaptic membrane / growth hormone secretion / synaptobrevin 2-SNAP-25-syntaxin-1a complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / presynaptic active zone membrane / synaptic vesicle fusion to presynaptic active zone membrane / SNARE complex assembly / synaptic vesicle priming / intracellular organelle / regulation of synapse assembly / calcium ion-regulated exocytosis of neurotransmitter / vesicle-fusing ATPase / positive regulation of hormone secretion / sleep / SNAP receptor activity / vesicle fusion / synaptic vesicle exocytosis / regulation of neuron projection development / positive regulation of receptor recycling / SNARE complex / syntaxin-1 binding / myosin binding / syntaxin binding / voltage-gated potassium channel activity / exocytosis / ionotropic glutamate receptor binding / long-term memory / positive regulation of insulin secretion / SNARE binding / voltage-gated potassium channel complex / axonal growth cone / axonogenesis / filopodium / endosomal transport / neuron differentiation / potassium ion transport / ATPase activity, coupled / PDZ domain binding / calcium-dependent protein binding / intracellular protein transport / positive regulation of protein catabolic process / terminal bouton / actin cytoskeleton / midbody / growth cone / lamellipodium / protein N-terminus binding / ion channel binding / ATPase activity / endosome / protein-containing complex binding / neuron projection / cell junction / myelin sheath / membrane raft / glutamatergic synapse / axon / protein domain specific binding / neuronal cell body / protein kinase binding / perinuclear region of cytoplasm / membrane / ATP binding / identical protein binding / plasma membrane / metal ion binding / cytosol / cytoplasm / Vesicle-fusing ATPase / Synaptosomal-associated protein 25
Function and homology information
Specimen sourceCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.8 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)541,98718
Polyers536,5687
Non-polymers5,41911
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 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: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 9 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (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, 20MULTIPLE SOURCES
2N-ethylmaleimide sensitive factorCOMPLEX11RECOMBINANT
3Synaptosomal-associated protein 25COMPLEX21RECOMBINANT
Molecular weight
IDValueEntity assembly IDExperimental value
10.628533 MDa1NO
20.496812 MDa1NO
30.023625 MDa1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
2210116Rattus norvegicus (Norway rat)
3310116Rattus norvegicus (Norway rat)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli (E. coli)
33562Escherichia 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 mesh size: 400 / 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 microns / 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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 184555 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 3J96

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