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- EMDB-9103: The D1 and D2 domain rings of NSF engaging the SNAP-25 N-terminus... -

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Basic information

Entry
Database: EMDB / ID: EMD-9103
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)
Map dataThe unsharpened map.
Sample
  • Complex: 20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
    • Complex: N-ethylmaleimide sensitive factor
      • Protein or peptide: Vesicle-fusing ATPase
    • Complex: Synaptosomal-associated protein 25SNAP25
      • Protein or peptide: Synaptosomal-associated protein 25SNAP25
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsSNARE / NSF / SNAP / ATPase / AAA / disassembly / synapse / membrane fusion / exocytosis / HYDROLASE
Function / homology
Function and homology information


BLOC-1 complex / SNARE complex disassembly / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling / extrinsic component of presynaptic membrane / 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 / Glutamate Neurotransmitter Release Cycle ...BLOC-1 complex / SNARE complex disassembly / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling / extrinsic component of presynaptic membrane / 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 / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / presynaptic dense core vesicle exocytosis / ribbon synapse / regulation of establishment of protein localization / calcium ion-regulated exocytosis of neurotransmitter / ATP-dependent protein disaggregase activity / SNARE complex / SNAP receptor activity / neurotransmitter secretion / positive regulation of hormone secretion / neurotransmitter receptor internalization / vesicle-fusing ATPase / SNARE complex assembly / syntaxin-1 binding / synaptic vesicle priming / Neutrophil degranulation / regulation of synapse assembly / endosomal transport / myosin binding / positive regulation of receptor recycling / regulation of neuron projection development / exocytosis / synaptic vesicle exocytosis / voltage-gated potassium channel activity / associative learning / long-term memory / voltage-gated potassium channel complex / axonal growth cone / presynaptic active zone membrane / somatodendritic compartment / ionotropic glutamate receptor binding / photoreceptor inner segment / axonogenesis / SNARE binding / locomotory behavior / filopodium / long-term synaptic potentiation / PDZ domain binding / intracellular protein transport / trans-Golgi network / potassium ion transport / terminal bouton / positive regulation of insulin secretion / neuron differentiation / positive regulation of protein catabolic process / calcium-dependent protein binding / synaptic vesicle / actin cytoskeleton / presynapse / presynaptic membrane / lamellipodium / cell cortex / growth cone / midbody / postsynapse / transmembrane transporter binding / cytoskeleton / endosome / neuron projection / axon / protein domain specific binding / neuronal cell body / lipid binding / glutamatergic synapse / synapse / protein-containing complex binding / protein kinase binding / perinuclear region of cytoplasm / Golgi apparatus / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / 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 ...Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Vesicle-fusing ATPase / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWhite KI / Zhao M
Funding support United States, 1 items
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, 2018-
Header (metadata) releaseSep 19, 2018-
Map releaseSep 19, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mdp
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9103.map.gz / Format: CCP4 / Size: 46.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe unsharpened map.
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0135 / Movie #1: 0.0135
Minimum - Maximum-0.009235976 - 0.043628827
Average (Standard dev.)0.00005939628 (±0.0024336001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions230230230
Spacing230230230
CellA=B=C: 301.3 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z230230230
origin x/y/z0.0000.0000.000
length x/y/z301.300301.300301.300
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS230230230
D min/max/mean-0.0090.0440.000

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Supplemental data

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Additional map: The sharpened map.

Fileemd_9103_additional.map
AnnotationThe sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : 20S supercomplex consisting of soluble neuronal SNARE complex, al...

EntireName: 20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
Components
  • Complex: 20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
    • Complex: N-ethylmaleimide sensitive factor
      • Protein or peptide: Vesicle-fusing ATPase
    • Complex: Synaptosomal-associated protein 25SNAP25
      • Protein or peptide: Synaptosomal-associated protein 25SNAP25
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: 20S supercomplex consisting of soluble neuronal SNARE complex, al...

SupramoleculeName: 20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 23.625 KDa

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Supramolecule #2: N-ethylmaleimide sensitive factor

SupramoleculeName: N-ethylmaleimide sensitive factor / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: Synaptosomal-associated protein 25

SupramoleculeName: Synaptosomal-associated protein 25 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Vesicle-fusing ATPase

MacromoleculeName: Vesicle-fusing ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Cricetulus griseus (Chinese hamster)
Molecular weightTheoretical: 85.509227 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHDY DIPTTENLYF QGAHMAGRSM QAARCPTDEL SLSNCAVVSE KDYQSGQHVI VRTSPNHKYI FTLRTHPSVV PGSVAFSLP QRKWAGLSIG QEIEVALYSF DKAKQCIGTM TIEIDFLQKK NIDSNPYDTD KMAAEFIQQF NNQAFSVGQQ L VFSFNDKL ...String:
MGHHHHHHDY DIPTTENLYF QGAHMAGRSM QAARCPTDEL SLSNCAVVSE KDYQSGQHVI VRTSPNHKYI FTLRTHPSVV PGSVAFSLP QRKWAGLSIG QEIEVALYSF DKAKQCIGTM TIEIDFLQKK NIDSNPYDTD KMAAEFIQQF NNQAFSVGQQ L VFSFNDKL FGLLVKDIEA MDPSILKGEP ASGKRQKIEV GLVVGNSQVA FEKAENSSLN LIGKAKTKEN RQSIINPDWN FE KMGIGGL DKEFSDIFRR AFASRVFPPE IVEQMGCKHV KGILLYGPPG CGKTLLARQI GKMLNAREPK VVNGPEILNK YVG ESEANI RKLFADAEEE QRRLGANSGL HIIIFDEIDA ICKQRGSMAG STGVHDTVVN QLLSKIDGVE QLNNILVIGM TNRP DLIDE ALLRPGRLEV KMEIGLPDEK GRLQILHIHT ARMRGHQLLS ADVDIKELAV ETKNFSGAEL EGLVRAAQST AMNRH IIAS TKVEVDMEKA ESLQVTRGDF LASLENDIKP AFGTNQEDYA SYIMNGIIKW GDPVTRVLDD GELLVQQTKN SDRTPL VSV LLEGPPHSGK TALAAKIAEE SNFPFIKICS PDKMIGFSET AKCQAMKKIF DDAYKSQLSC VVVDDIERLL DYVPIGP RF SNLVLQALLV LLKKAPPQGR KLLIIGTTSR KDVLQEMEML NAFSTTIHVP NIATGEQLLE ALELLGNFKD KERTTIAQ Q VKGKKVWIGI KKLLMLIEMS LQMDPEYRVR KFLALLREEG ASPLDFD

UniProtKB: Vesicle-fusing ATPase

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Macromolecule #2: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 23.512387 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASMAEDADM RNELEEMQRR ADQLADESLE STRRMLQLVE ESKDAGIRTL VMLDEQGEQL DRVEEGMNHI NQDMKEAEKN LKDLGKCCG LFICPCNKLK SSDAYKKAWG NNQDGVVASQ PARVVDEREQ MAISGGFIRR VTNDARENEM DENLEQVSGI I GNLRHMAL ...String:
MASMAEDADM RNELEEMQRR ADQLADESLE STRRMLQLVE ESKDAGIRTL VMLDEQGEQL DRVEEGMNHI NQDMKEAEKN LKDLGKCCG LFICPCNKLK SSDAYKKAWG NNQDGVVASQ PARVVDEREQ MAISGGFIRR VTNDARENEM DENLEQVSGI I GNLRHMAL DMGNEIDTQN RQIDRIMEKA DSNKTRIDEA NQRATKMLG

UniProtKB: Synaptosomal-associated protein 25

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 9 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration15 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
0.05 MC4H11NO3Tris HCl
0.15 MNaClSodium chlorideSodium chloride
0.001 MC10H16N5O13P3Adenosine triphosphate
0.001 MC10H16N2O8Ethylenediaminetetraacetic acid
0.001 MC4H10O2S2Dithiothreitol
0.05 % v/vNonidet P-40
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK I / Details: Blot for 3.5 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 2-40 / Number grids imaged: 2 / Number real images: 5418 / Average exposure time: 10.0 sec. / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 475680
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 184555
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6mdp:
The 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)

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