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- EMDB-9102: The D1 and D2 domain rings of NSF engaging the SNAP-25 N-terminus... -

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Entry
Database: EMDB / ID: 9102
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 1)
Map dataUnsharpened map.
Sample20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
  • N-ethylmaleimide sensitive factor
  • (Synaptosomal-associated protein ...) x 2
  • Vesicle-fusing ATPase
  • (ligand) x 2
Function / homologyAspartate decarboxylase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / CDC48 domain 2-like superfamily / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / Synaptosomal-associated protein 25 / Vesicle-fusing ATPase / CDC48, N-terminal subdomain / SNAP-25 ...Aspartate decarboxylase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / CDC48 domain 2-like superfamily / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / Synaptosomal-associated protein 25 / Vesicle-fusing ATPase / CDC48, N-terminal subdomain / SNAP-25 / Target SNARE coiled-coil homology domain / CDC48, domain 2 / 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. / 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 / synaptic vesicle fusion to presynaptic active zone membrane / presynaptic active zone membrane / SNARE complex assembly / synaptic vesicle priming / intracellular organelle / calcium ion-regulated exocytosis of neurotransmitter / regulation of synapse assembly / vesicle-fusing ATPase / vesicle fusion / positive regulation of hormone secretion / SNAP receptor activity / sleep / synaptic vesicle exocytosis / regulation of neuron projection development / SNARE complex / positive regulation of receptor recycling / 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 / potassium ion transport / ATPase activity, coupled / neuron differentiation / PDZ domain binding / positive regulation of protein catabolic process / terminal bouton / calcium-dependent protein binding / intracellular protein transport / actin cytoskeleton / midbody / growth cone / lamellipodium / protein N-terminus binding / ion channel binding / ATPase activity / endosome / protein-containing complex binding / cell junction / neuron projection / membrane raft / myelin sheath / axon / glutamatergic synapse / 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
SourceRattus norvegicus (Norway rat) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsWhite KI / Zhao M / Brunger AT
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 ReportPDB-ID: 6mdo

SummaryFull reportAbout validation report
DateDeposition: Sep 4, 2018 / Header (metadata) release: Sep 19, 2018 / Map release: Sep 19, 2018 / Last update: Sep 26, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
  • Download
  • 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-6mdo
  • 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

Fileemd_9102.map.gz (map file in CCP4 format, 48669 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
230 pix
1.31 Å/pix.
= 301.3 Å
230 pix
1.31 Å/pix.
= 301.3 Å
230 pix
1.31 Å/pix.
= 301.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density
Contour Level:0.0135 (by author), 0.0135 (movie #1):
Minimum - Maximum-0.15603079 - 0.28024182
Average (Standard dev.)0.00017620984 (0.010243639)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions230230230
Origin0.00.00.0
Limit229.0229.0229.0
Spacing230230230
CellA=B=C: 301.3 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS230230230
D min/max/mean-0.0100.0460.000

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Supplemental data

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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)
Number of components: 7

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Component #1: protein, 20S supercomplex consisting of soluble neuronal SNARE co...

ProteinName: 20S supercomplex consisting of soluble neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
Recombinant expression: No
MassTheoretical: 23.625 kDa

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Component #2: protein, N-ethylmaleimide sensitive factor

ProteinName: N-ethylmaleimide sensitive factor / Recombinant expression: No
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Synaptosomal-associated protein 25

ProteinName: Synaptosomal-associated protein 25SNAP25 / Recombinant expression: No
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Vesicle-fusing ATPase

ProteinName: Vesicle-fusing ATPase / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 85.509227 kDa
SourceSpecies: Cricetulus griseus (Chinese hamster)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Synaptosomal-associated protein 25

ProteinName: Synaptosomal-associated protein 25SNAP25 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.512387 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 9 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Component #7: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 15 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 % / Details: Blot for 3.5 seconds before plunging..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 58 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5418

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 166620
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION
CTF correction: CTF correction was carried out in Relion with reconstruction step.
Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3J96
Output model

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