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- EMDB-2013: Electron microscopy negative staining map of the cross-linked p97... -

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Basic information

Entry
Database: EMDB / ID: EMD-2013
TitleElectron microscopy negative staining map of the cross-linked p97-Ufd1-Npl4 complex
Map dataSurface rendered p97-ATPase in complex with the adaptor Ufd1-Npl4
Sample
  • Sample: p97-Ufd1-Npl4 cross-linked with glutaraldehyde
  • Protein or peptide: p97
  • Protein or peptide: Ufd1
  • Protein or peptide: Npl4
KeywordsEM / p97 / Ufd1-Npl4 / asymmetric complex / ATPase
Biological speciesMus musculus (house mouse) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 28.0 Å
AuthorsBebeacua C / Forster A / McKeown C / Meyer HH / Zhang X / Freemont PS
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy.
Authors: Cecilia Bebeacua / Andreas Förster / Ciarán McKeown / Hemmo H Meyer / Xiaodong Zhang / Paul S Freemont /
Abstract: p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how ...p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.
History
DepositionDec 23, 2011-
Header (metadata) releaseJan 13, 2012-
Map releaseJan 13, 2012-
UpdateJun 10, 2015-
Current statusJun 10, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd2013fig.png

Downloads & links

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Map

FileDownload / File: emd_2013.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSurface rendered p97-ATPase in complex with the adaptor Ufd1-Npl4
Voxel sizeX=Y=Z: 3.53 Å
Density
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-1.68775 - 13.483599999999999
Average (Standard dev.)0.115421 (±0.819604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 451.84 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.533.533.53
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z451.840451.840451.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-1.68813.4840.115

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Supplemental data

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Sample components

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Entire : p97-Ufd1-Npl4 cross-linked with glutaraldehyde

EntireName: p97-Ufd1-Npl4 cross-linked with glutaraldehyde
Components
  • Sample: p97-Ufd1-Npl4 cross-linked with glutaraldehyde
  • Protein or peptide: p97
  • Protein or peptide: Ufd1
  • Protein or peptide: Npl4

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Supramolecule #1000: p97-Ufd1-Npl4 cross-linked with glutaraldehyde

SupramoleculeName: p97-Ufd1-Npl4 cross-linked with glutaraldehyde / type: sample / ID: 1000
Oligomeric state: One hexamer of p97 binds to one Ufd1-Npl4 dimer
Number unique components: 3
Molecular weightExperimental: 623 KDa / Theoretical: 623 KDa

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Macromolecule #1: p97

MacromoleculeName: p97 / type: protein_or_peptide / ID: 1 / Name.synonym: p97 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta

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Macromolecule #2: Ufd1

MacromoleculeName: Ufd1 / type: protein_or_peptide / ID: 2 / Name.synonym: Ufd1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta

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Macromolecule #3: Npl4

MacromoleculeName: Npl4 / type: protein_or_peptide / ID: 3 / Name.synonym: Npl4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway rat / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8 / Details: 25 mM HEPES, 500 mM KCl
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 60 seconds.
GridDetails: 200 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder: RT / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJul 1, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Number real images: 1000 / Average electron dose: 10 e/Å2

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: OTHER / Software - Name: IMAGIC / Number images used: 15000

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