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- EMDB-2583: S. cerevisiae Pex1/6 wild type complex bound to gammaS ATP -

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Basic information

Entry
Database: EMDB / ID: EMD-2583
TitleS. cerevisiae Pex1/6 wild type complex bound to gammaS ATP
Map datanegative stain reconstruction of wild type Pex1/6 complex bound to gammaS ATP
Sample
  • Sample: S. cerevisiae Pex1/Pex6 wild type complex
  • Protein or peptide: peroxisomal biogenesis factor 1 (Pex1)
  • Protein or peptide: peroxisomal biogenesis factor 6 (Pex6)
KeywordsType-II AAA+ protein complex / heterohexamer / peroxisomal biogenesis
Function / homology
Function and homology information


protein import into peroxisome matrix, receptor recycling / protein import into peroxisome matrix / protein transporter activity / peroxisomal membrane / ATPase complex / protein unfolding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / peroxisome / ATP hydrolysis activity / ATP binding / cytosol
Similarity search - Function
Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like superfamily / : / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) ...Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like superfamily / : / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Peroxisomal ATPase PEX1 / Peroxisomal ATPase PEX6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsCiniawsky S / Grimm I / Saffian D / Girzalsky W / Erdmann R / Wendler P
CitationJournal: Nat Commun / Year: 2015
Title: Molecular snapshots of the Pex1/6 AAA+ complex in action.
Authors: Susanne Ciniawsky / Immanuel Grimm / Delia Saffian / Wolfgang Girzalsky / Ralf Erdmann / Petra Wendler /
Abstract: The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans ...The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential substrate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA+ assemblies.
History
DepositionFeb 19, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseJul 1, 2015-
UpdateJul 1, 2015-
Current statusJul 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_2583.tif

Downloads & links

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Map

FileDownload / File: emd_2583.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative stain reconstruction of wild type Pex1/6 complex bound to gammaS ATP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.9 Å/pix.
x 160 pix.
= 464. Å		2.9 Å/pix.
x 160 pix.
= 464. Å		2.9 Å/pix.
x 160 pix.
= 464. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-5.24901199 - 9.90154457
Average (Standard dev.)0.01875458 (±0.34628552)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 464.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z464.000464.000464.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-5.2499.9020.019

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Supplemental data

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Sample components

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Entire : S. cerevisiae Pex1/Pex6 wild type complex

EntireName: S. cerevisiae Pex1/Pex6 wild type complex
Components
  • Sample: S. cerevisiae Pex1/Pex6 wild type complex
  • Protein or peptide: peroxisomal biogenesis factor 1 (Pex1)
  • Protein or peptide: peroxisomal biogenesis factor 6 (Pex6)

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Supramolecule #1000: S. cerevisiae Pex1/Pex6 wild type complex

SupramoleculeName: S. cerevisiae Pex1/Pex6 wild type complex / type: sample / ID: 1000
Details: heterohexamer purified and assembled in the presence of nucleotide
Oligomeric state: heterohexamer / Number unique components: 2
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa / Method: gel filtration

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Macromolecule #1: peroxisomal biogenesis factor 1 (Pex1)

MacromoleculeName: peroxisomal biogenesis factor 1 (Pex1) / type: protein_or_peptide / ID: 1 / Name.synonym: Pas1 / Number of copies: 3 / Oligomeric state: heterohexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: peroxisome / Location in cell: cytosol
Molecular weightExperimental: 117 KDa / Theoretical: 117 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Tuner / Recombinant plasmid: pGEX-4T-2
SequenceUniProtKB: Peroxisomal ATPase PEX1

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Macromolecule #2: peroxisomal biogenesis factor 6 (Pex6)

MacromoleculeName: peroxisomal biogenesis factor 6 (Pex6) / type: protein_or_peptide / ID: 2 / Name.synonym: Pas8 / Number of copies: 3 / Oligomeric state: heterohexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: peroxisome / Location in cell: cytosol
Molecular weightExperimental: 116 KDa / Theoretical: 116 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Tuner / Recombinant plasmid: pETDuet-1
SequenceUniProtKB: Peroxisomal ATPase PEX6

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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: 7.5
Details: 20mM Tris-HCl, 20mM NaCl, 10mM MgCl2, 2.5 mM gammaS ATP
StainingType: NEGATIVE
Details: Grid with adsorbed protein was stained with 2% uranyl acetate for 15 seconds and blotted to near dryness.
GridDetails: 400 Cu mesh continuous carbon grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
DateSep 26, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 30 µm / Number real images: 94 / Average electron dose: 18 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.35 µm / Nominal magnification: 103448
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping of micrographs
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: MRC, IMAGIC, SPIDER / Number images used: 3895
Final two d classificationNumber classes: 500

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