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- EMDB-9102: 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-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 data
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 / homology
Function and homology information


SNARE complex disassembly / anchored component of presynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a complex / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / growth hormone secretion / 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 / short-term synaptic potentiation ...SNARE complex disassembly / anchored component of presynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a complex / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / growth hormone secretion / 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 / short-term synaptic potentiation / regulation of establishment of protein localization / calcium ion-regulated exocytosis of neurotransmitter / neurotransmitter receptor internalization / intracellular organelle / vesicle-fusing ATPase / positive regulation of hormone secretion / SNARE complex / vesicle fusion / SNAP receptor activity / SNARE complex assembly / synaptic vesicle priming / positive regulation of synaptic plasticity / regulation of synapse assembly / positive regulation of receptor recycling / sleep / syntaxin-1 binding / synaptic vesicle exocytosis / neurotransmitter secretion / myosin binding / regulation of neuron projection development / syntaxin binding / ionotropic glutamate receptor binding / exocytosis / voltage-gated potassium channel activity / associative learning / long-term memory / positive regulation of insulin secretion / voltage-gated potassium channel complex / somatodendritic compartment / SNARE binding / long-term synaptic potentiation / axonal growth cone / axonogenesis / photoreceptor inner segment / locomotory behavior / endosomal transport / filopodium / potassium ion transport / go:0042623: / PDZ domain binding / neuron differentiation / positive regulation of protein catabolic process / presynaptic membrane / extrinsic component of cytoplasmic side of plasma membrane / trans-Golgi network / intracellular protein transport / terminal bouton / midbody / postsynapse / actin cytoskeleton / calcium-dependent protein binding / growth cone / lamellipodium / synaptic vesicle / protein N-terminus binding / ion channel binding / endosome / cytoskeleton / ATPase activity / synapse / neuron projection / cell junction / membrane raft / glutamatergic synapse / axon / protein domain specific binding / neuronal cell body / protein-containing complex binding / protein kinase binding / perinuclear region of cytoplasm / membrane / ATP binding / identical protein binding / plasma membrane / metal ion binding / cytosol / cytoplasm
AAA+ ATPase domain / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core ...AAA+ ATPase domain / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / Target SNARE coiled-coil homology domain / CDC48, N-terminal subdomain / SNAP-25 domain
Vesicle-fusing ATPase / Synaptosomal-associated protein 25
Biological speciesRattus norvegicus (Norway rat) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsWhite KI / Zhao M / Brunger AT
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.
Validation ReportPDB-ID: 6mdo

SummaryFull reportAbout validation report
History
DepositionSep 4, 2018-
Header (metadata) releaseSep 19, 2018-
Map releaseSep 19, 2018-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing 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
  • 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

FileDownload / File: emd_9102.map.gz / Format: CCP4 / Size: 46.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 230 pix.
= 301.3 Å
1.31 Å/pix.
x 230 pix.
= 301.3 Å
1.31 Å/pix.
x 230 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 LevelBy AUTHOR: 0.0135 / Movie #1: 0.0135
Minimum - Maximum-0.15603079 - 0.28024182
Average (Standard dev.)0.00017620984 (±0.010243639)
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-0.0100.0460.000

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Supplemental data

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Additional map: Sharpened map.

Fileemd_9102_additional.map
AnnotationSharpened 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)
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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