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- EMDB-1059: Electron cryomicroscopy structure of N-ethyl maleimide sensitive ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1059
TitleElectron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution.
Map data3D density map of NSF?alpha-SNAP?SNARE (20S)
Sample
  • Sample: NSF alpha-SNAP SNARE 20S
  • Protein or peptide: N-ethyl Maleimide Sensitive Factor
  • Protein or peptide: Soluble NSF Attachment Protein
  • Protein or peptide: Soluble NSF Attachment Protein Receptor
Biological speciesCricetulus griseus (Chinese hamster) / Bos taurus (cattle) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 11.0 Å
AuthorsFurst J / Sutton RB / Chen J / Brunger AT / Grigorieff N
CitationJournal: EMBO J / Year: 2003
Title: Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution.
Authors: Johannes Furst / R Bryan Sutton / James Chen / Axel T Brunger / Nikolaus Grigorieff /
Abstract: N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We ...N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.
History
DepositionOct 10, 2003-
Header (metadata) releaseOct 10, 2003-
Map releaseOct 10, 2003-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.736926654
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.736926654
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1059.gif

Downloads & links

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Map

FileDownload / File: emd_1059.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D density map of NSF?alpha-SNAP?SNARE (20S)
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour Level1: 0.144 / Movie #1: 0.7369267
Minimum - Maximum-0.681735 - 1.45625
Average (Standard dev.)0.0257073 (±0.145834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin111
Dimensions909090
Spacing909090
CellA=B=C: 315 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z315.000315.000315.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0052
NX/NY/NZ12812855
MAP C/R/S123
start NC/NR/NS111
NC/NR/NS909090
D min/max/mean-0.6821.4560.026

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Supplemental data

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Sample components

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Entire : NSF alpha-SNAP SNARE 20S

EntireName: NSF alpha-SNAP SNARE 20S
Components
  • Sample: NSF alpha-SNAP SNARE 20S
  • Protein or peptide: N-ethyl Maleimide Sensitive Factor
  • Protein or peptide: Soluble NSF Attachment Protein
  • Protein or peptide: Soluble NSF Attachment Protein Receptor

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Supramolecule #1000: NSF alpha-SNAP SNARE 20S

SupramoleculeName: NSF alpha-SNAP SNARE 20S / type: sample / ID: 1000
Details: The sample was monodisperse. Some domains were disordered.
Number unique components: 3
Molecular weightExperimental: 680 KDa / Method: gel permeation chromatography

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Macromolecule #1: N-ethyl Maleimide Sensitive Factor

MacromoleculeName: N-ethyl Maleimide Sensitive Factor / type: protein_or_peptide / ID: 1 / Name.synonym: NSF / Details: 6 subunits / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Cricetulus griseus (Chinese hamster) / synonym: Chinese Hamster
Molecular weightExperimental: 82.8 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: Soluble NSF Attachment Protein

MacromoleculeName: Soluble NSF Attachment Protein / type: protein_or_peptide / ID: 2 / Name.synonym: alpha-SNAP / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle
Molecular weightExperimental: 33.4 KDa

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Macromolecule #3: Soluble NSF Attachment Protein Receptor

MacromoleculeName: Soluble NSF Attachment Protein Receptor / type: protein_or_peptide / ID: 3 / Name.synonym: SNARE / Recombinant expression: Yes
Source (natural)Organism: unidentified (others) / synonym: Chinese Hamster
Molecular weightExperimental: 82.3 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
Details: 20mM HEPES, pH 7.4, 300 mM NACl, 2 mM MgCl2, 5mM b-mercaptoethanol, 0.01/0.1 mM ADP/ATP nucleotide
StainingType: NEGATIVE / Details: cryo (no stain)
GridDetails: holey carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 85 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Technion plunger

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 85 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Average electron dose: 10 e/Å2 / Details: 3 x 3 averaging, final step size = 27 microns / Od range: 1.4 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: FREALIGN
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, FREALIGN / Details: Final map was calculated from three data sets / Number images used: 31592
Detailsmanual particle selection

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