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- EMDB-6359: Cryo-EM structure of the peroxisomal Pex1/Pex6 complex in ATP-gam... -

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Entry
Database: EMDB / ID: EMD-6359
TitleCryo-EM structure of the peroxisomal Pex1/Pex6 complex in ATP-gamma-S state
Map dataReconstruction of Pex1/Pex6 AAA ATPase complex in ATP-gamma-S using single-particle cryo-electron microscopy without imposed symmetry
Sample
  • Sample: Peroxisomal Pex1/Pex6 ATPase complex
  • Protein or peptide: Peroxisomal ATPase Pex1
  • Protein or peptide: Peroxisomal ATPase Pex6
KeywordsAAA ATPase / cryoelectron microscopy / peroxisome
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 / 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 / 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 / cryo EM / Resolution: 7.2 Å
AuthorsBlok NB / Tan D / Wang RY / Penczek PA / Baker D / DiMaio F / Rapoport TA / Walz T
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy.
Authors: Neil B Blok / Dongyan Tan / Ray Yu-Ruei Wang / Pawel A Penczek / David Baker / Frank DiMaio / Tom A Rapoport / Thomas Walz /
Abstract: Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the ...Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.
History
DepositionJun 20, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseJul 22, 2015-
UpdateMay 4, 2016-
Current statusMay 4, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6359.gif

Downloads & links

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Map

FileDownload / File: emd_6359.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Pex1/Pex6 AAA ATPase complex in ATP-gamma-S using single-particle cryo-electron microscopy without imposed symmetry
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy EMDB: 0.0123 / Movie #1: 0.012
Minimum - Maximum-0.00397698 - 0.02711316
Average (Standard dev.)-0.000012 (±0.00245146)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 297.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z297.600297.600297.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.0040.027-0.000

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Supplemental data

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Sample components

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Entire : Peroxisomal Pex1/Pex6 ATPase complex

EntireName: Peroxisomal Pex1/Pex6 ATPase complex
Components
  • Sample: Peroxisomal Pex1/Pex6 ATPase complex
  • Protein or peptide: Peroxisomal ATPase Pex1
  • Protein or peptide: Peroxisomal ATPase Pex6

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Supramolecule #1000: Peroxisomal Pex1/Pex6 ATPase complex

SupramoleculeName: Peroxisomal Pex1/Pex6 ATPase complex / type: sample / ID: 1000
Details: The sample was freshly prepared before being loaded onto grids.
Oligomeric state: heterohexamer formed by three Pex1 and three Pex6
Number unique components: 2
Molecular weightTheoretical: 726 KDa

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Macromolecule #1: Peroxisomal ATPase Pex1

MacromoleculeName: Peroxisomal ATPase Pex1 / type: protein_or_peptide / ID: 1 / Name.synonym: Pex1 / Number of copies: 3
Oligomeric state: Three molecules of Pex1 interact with three molecules of Pex6 to form a heterohexamer of alternating subunits.
Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Organelle: peroxisome / Location in cell: cytoplasm
Molecular weightTheoretical: 122 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: InvSc1 / Recombinant plasmid: pRS423 Gal1
SequenceUniProtKB: Peroxisomal ATPase PEX1 / InterPro: Peroxisome biogenesis factor 1

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Macromolecule #2: Peroxisomal ATPase Pex6

MacromoleculeName: Peroxisomal ATPase Pex6 / type: protein_or_peptide / ID: 2 / Name.synonym: Pex6 / Number of copies: 3
Oligomeric state: Three molecules of Pex1 interact with three molecules of Pex6 to form a heterohexamer of alternating subunits.
Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Organelle: peroxisome / Location in cell: cytoplasm
Molecular weightTheoretical: 120 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: InvSc1 / Recombinant plasmid: pRS424 Gal1
SequenceUniProtKB: Peroxisomal ATPase PEX6

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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

Concentration0.6 mg/mL
BufferpH: 7.5
Details: 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1mM DTT, 0.3 mM ATP-gamma-S
GridDetails: Quantifoil 400 mesh holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40410 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: -2.2 µm / Nominal defocus min: -0.9 µm / Nominal magnification: 29000
Sample stageSpecimen holder: Oxford instrument nitrogen-cooled side-entry holder
Specimen holder model: GATAN LIQUID NITROGEN
DetailsImages were recorded using a Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
DateMay 13, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 1169 / Average electron dose: 38 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: OTHER / Software - Name: SPARX, RELION
Details: Initial model building was done in SPARX using the common-line method. 3D classification, refinement, and subsequent reconstruction were performed using RELION.
Number images used: 46655

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