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- EMDB-2874: Cryo electron microscopy of SNAP-SNARE assembly in 20S particle -

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Basic information

Entry
Database: EMDB / ID: EMD-2874
TitleCryo electron microscopy of SNAP-SNARE assembly in 20S particle
Map dataReconstruction of alpha-SNAP-SNARE assembly in 20S particle
Sample
  • Sample: alpha-SNAP-SNARE assembly in 20S particle
  • Protein or peptide: soluble NSF attachment protein receptor
  • Protein or peptide: alpha soluble NSF attachment protein
Keywords20S particles / SNARE / alpha-SNAP / membrane fusion
Biological speciesRattus norvegicus (Norway rat) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.35 Å
AuthorsZhou Q / Huang X / Sun S / Li XM / Wang HW / Sui SF
CitationJournal: Cell Res / Year: 2015
Title: Cryo-EM structure of SNAP-SNARE assembly in 20S particle.
Authors: Qiang Zhou / Xuan Huang / Shan Sun / Xueming Li / Hong-Wei Wang / Sen-Fang Sui /
Abstract: N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after ...N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and α-SNAP, we performed single-particle cryo-electron microscopy analysis of 20S particles and determined the structure of the α-SNAP-SNARE assembly portion at a resolution of 7.35 Å. The structure illustrates that four α-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each α-SNAP forms a chock protruding into the groove of the SNARE four-helix bundle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four α-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.
History
DepositionJan 30, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseMay 6, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2874.png

Downloads & links

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Map

FileDownload / File: emd_2874.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of alpha-SNAP-SNARE assembly in 20S particle
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.0347008 - 0.07660946
Average (Standard dev.)0.00060082 (±0.0071753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 168.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z168.960168.960168.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0350.0770.001

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Supplemental data

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Segmentation: This is a soft spherical mask.

AnnotationThis is a soft spherical mask.
Fileemd_2874_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : alpha-SNAP-SNARE assembly in 20S particle

EntireName: alpha-SNAP-SNARE assembly in 20S particle
Components
  • Sample: alpha-SNAP-SNARE assembly in 20S particle
  • Protein or peptide: soluble NSF attachment protein receptor
  • Protein or peptide: alpha soluble NSF attachment protein

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Supramolecule #1000: alpha-SNAP-SNARE assembly in 20S particle

SupramoleculeName: alpha-SNAP-SNARE assembly in 20S particle / type: sample / ID: 1000 / Details: 20S particle was prepared in nanodisc.
Oligomeric state: One homotetramer of alpha-SNAP binds to one monomer of SNARE complex.
Number unique components: 2
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: soluble NSF attachment protein receptor

MacromoleculeName: soluble NSF attachment protein receptor / type: protein_or_peptide / ID: 1 / Name.synonym: SNARE / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway Rat
Molecular weightTheoretical: 66 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: alpha soluble NSF attachment protein

MacromoleculeName: alpha soluble NSF attachment protein / type: protein_or_peptide / ID: 2 / Name.synonym: alpha-SNAP / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: cattle
Molecular weightTheoretical: 33 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.2 mg/mL
GridDetails: 400 mesh copper grid with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 second before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 22,500 times magnification.
DateJul 2, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2349 / Average electron dose: 46 e/Å2
Details: Every image is the average of motion-corrected movie frames.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.35 Å / Resolution method: OTHER / Software - Name: relion, 1.3 / Number images used: 64710
DetailsThe particles were automatic selected.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1sfc


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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