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- EMDB-6255: Negative stain reconstruction of the Pex1/Pex6 complex in presenc... -

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Basic information

Entry
Database: EMDB / ID: EMD-6255
TitleNegative stain reconstruction of the Pex1/Pex6 complex in presence of ATP
Map dataNegative stain reconstruction of the Pex1/Pex6 complex in presence of 3 mM ATP
Sample
  • Sample: Pex1/Pex6 complex in presence of 3 mM ATP
  • Protein or peptide: Peroxisomal ATPase PEX1
  • Protein or peptide: Peroxisomal ATPase PEX6
KeywordsPeroxisome / AAA+ ATPase / Pex1 / Pex6
Function / homology
Function and homology information


ATP hydrolysis activity => GO:0016887 / : / protein targeting to peroxisome / protein import into peroxisome matrix, receptor recycling / protein import into peroxisome matrix / protein binding / peroxisome organization / protein transporter activity / peroxisomal membrane / ATPase complex ...ATP hydrolysis activity => GO:0016887 / : / protein targeting to peroxisome / protein import into peroxisome matrix, receptor recycling / protein import into peroxisome matrix / protein binding / peroxisome organization / 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 / protein heterodimerization activity / nucleotide binding / ATP hydrolysis activity / ATP binding / membrane / cytosol / cytoplasm
Similarity search - Function
Peroxisome biogenesis factor 1 / Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like superfamily / CDC48 domain 2-like superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site ...Peroxisome biogenesis factor 1 / Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like superfamily / CDC48 domain 2-like superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / 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: 23.4 Å
AuthorsGardner BM / Chowdhury S / Lander GC / Martin A
CitationJournal: J Mol Biol / Year: 2015
Title: The Pex1/Pex6 complex is a heterohexameric AAA+ motor with alternating and highly coordinated subunits.
Authors: Brooke M Gardner / Saikat Chowdhury / Gabriel C Lander / Andreas Martin /
Abstract: Pex1 and Pex6 are Type-2 AAA+ ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis ...Pex1 and Pex6 are Type-2 AAA+ ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here, we show that the ATP-dependent complex of Pex1 and Pex6 from Saccharomyces cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes, inhibits the ATP-hydrolysis activity of Pex1/Pex6.
History
DepositionJan 23, 2015-
Header (metadata) releaseFeb 18, 2015-
Map releaseFeb 18, 2015-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.56
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.56
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6255.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of the Pex1/Pex6 complex in presence of 3 mM ATP
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 1.56 / Movie #1: 1.56
Minimum - Maximum-7.98085403 - 8.27610874
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-7.9818.276-0.000

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Supplemental data

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Sample components

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Entire : Pex1/Pex6 complex in presence of 3 mM ATP

EntireName: Pex1/Pex6 complex in presence of 3 mM ATP
Components
  • Sample: Pex1/Pex6 complex in presence of 3 mM ATP
  • Protein or peptide: Peroxisomal ATPase PEX1
  • Protein or peptide: Peroxisomal ATPase PEX6

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Supramolecule #1000: Pex1/Pex6 complex in presence of 3 mM ATP

SupramoleculeName: Pex1/Pex6 complex in presence of 3 mM ATP / type: sample / ID: 1000
Details: 22 nM Pex1/Pex6 complex in presence of 3 mM ATP was used for negative stain electron microscopy. The sample was monodisperse.
Oligomeric state: Heterohexamer containing three copies of Pex1 and three copies of Pex6
Number unique components: 2
Molecular weightTheoretical: 707.076 KDa

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Macromolecule #1: Peroxisomal ATPase PEX1

MacromoleculeName: Peroxisomal ATPase PEX1 / type: protein_or_peptide / ID: 1
Name.synonym: Peroxin-1, Peroxisomal assembly protein 1, Peroxisome biogenesis protein 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, cytosol / Location in cell: peroxisome membrane
Molecular weightExperimental: 117 KDa / Theoretical: 117 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pCOLA-DUET
SequenceUniProtKB: Peroxisomal ATPase PEX1
GO: nucleotide binding, protein binding, ATP binding, cytoplasm, peroxisome, peroxisomal membrane, protein targeting to peroxisome, peroxisome organization, membrane, protein import into peroxisome ...GO: nucleotide binding, protein binding, ATP binding, cytoplasm, peroxisome, peroxisomal membrane, protein targeting to peroxisome, peroxisome organization, membrane, protein import into peroxisome matrix, receptor recycling, ATP hydrolysis activity, ATP hydrolysis activity => GO:0016887, protein heterodimerization activity
InterPro: AAA+ ATPase domain, ATPase, AAA-type, core, ATPase, AAA-type, conserved site, CDC48 domain 2-like superfamily, P-loop containing nucleoside triphosphate hydrolase, Peroxisome biogenesis ...InterPro: AAA+ ATPase domain, ATPase, AAA-type, core, ATPase, AAA-type, conserved site, CDC48 domain 2-like superfamily, P-loop containing nucleoside triphosphate hydrolase, Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold, 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: Peroxin-6 Peroxisomal assembly protein 8, Peroxisome biosynthesis protein 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, cytosol / Location in cell: peroxisome membrane
Molecular weightExperimental: 115 KDa / Theoretical: 115 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET-DUET
SequenceUniProtKB: Peroxisomal ATPase PEX6
GO: nucleotide binding, GO: 0001302, protein binding, ATP binding, cytoplasm, peroxisome, peroxisomal membrane, cytosol, peroxisome organization, membrane, protein import into peroxisome matrix, ...GO: nucleotide binding, GO: 0001302, protein binding, ATP binding, cytoplasm, peroxisome, peroxisomal membrane, cytosol, peroxisome organization, membrane, protein import into peroxisome matrix, receptor recycling, ATP hydrolysis activity, protein heterodimerization activity
InterPro: AAA+ ATPase domain, ATPase, AAA-type, core, ATPase, AAA-type, conserved site, P-loop containing nucleoside triphosphate hydrolase

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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

Concentration0.016 mg/mL
BufferpH: 7.6
Details: 60 mM HEPES, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 1 mM TCEP, 3 mM ATP
StainingType: NEGATIVE
Details: 4 microliters of sample was applied to a freshly plasma-cleaned thin carbon surface that was pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removing excess protein, negative ...Details: 4 microliters of sample was applied to a freshly plasma-cleaned thin carbon surface that was pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removing excess protein, negative staining was performed with 2% w/v uranyl formate solution.
GridDetails: 400 mesh Cu-Rh Maxtaform grid with a thin continuous carbon film on top
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.20 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 52000
Sample stageSpecimen holder: Room temperature side entry holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 294 K / Max: 297 K / Average: 295 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected using a quadrupole stigmator at 52,000 times magnification.
DetailsData were collected at room temperature.
DateNov 26, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 825 / Average electron dose: 20 e/Å2
Details: Data collected with the Leginon automated image acquisition software.
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping of whole micrograph
Final angle assignmentDetails: Theta 45 degrees, phi 45 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.4 Å / Resolution method: OTHER / Software - Name: Relion
Details: The amplitudes of the final reconstruction were adjusted using SPIDER to fit the density Fourier amplitudes to an experimental 1D low-angle X-ray scattering curve, then low-pass filtered.
Number images used: 11946
DetailsProcessing leading up to 3D reconstruction was performed using the Appion package. Particles were selected using the Difference of Gaussians (DoG)-based automated particle picker from micrographs. The stack was subjected to five iterations of iterative 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). The clean particle stack was subjected to twenty five iterations of 3D classification with five classes using the Relion suite. Particles belonging to well-resolved 3D class averages were used for further refinement by projection matching in Relion.
FSC plot (resolution estimation)

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