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- EMDB-7005: Negative stain reconstruction of the peroxisomal AAA-ATPase Pex1/... -

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Basic information

Entry
Database: EMDB / ID: EMD-7005
TitleNegative stain reconstruction of the peroxisomal AAA-ATPase Pex1/Pex6 complex associated with substrate Pex15
Map dataNegative stain reconstruction of the Pex15 bound Pex1-Pex6 complex.
Sample
  • Complex: Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 23.2 Å
AuthorsChowdhury S / Gardner BM / Castanzo DT / Stjepanovic G / Stefely MS / Hurley JH / Martin A / Lander GC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM094497, NSF-MCB- 1150288, DP2 EB020402-01, K99GM121880 United States
Howard Hughes Medical Institute (HHMI) United States
Department of Energy (DOE, United States) United States
CitationJournal: Nat Commun / Year: 2018
Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading.
Authors: Brooke M Gardner / Dominic T Castanzo / Saikat Chowdhury / Goran Stjepanovic / Matthew S Stefely / James H Hurley / Gabriel C Lander / Andreas Martin /
Abstract: Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored ...Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.
History
DepositionSep 1, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 20, 2017-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7005.png

Downloads & links

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Map

FileDownload / File: emd_7005.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of the Pex15 bound Pex1-Pex6 complex.
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 3.06 / Movie #1: 3.06
Minimum - Maximum-8.168757 - 10.292679
Average (Standard dev.)-0.048110332 (±1.2685611)
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-51-35-11
NX/NY/NZ11110799
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-8.16910.293-0.048

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Supplemental data

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Sample components

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Entire : Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15

EntireName: Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15
Components
  • Complex: Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15

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Supramolecule #1: Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15

SupramoleculeName: Complex between Pex1-Pex6 AAA-ATPase with substrate Pex15
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
Molecular weightTheoretical: 750 KDa

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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

BufferpH: 7.6
Details: 60 mM HEPES pH 7.6, 50 mM NaCl, 50 mM KCl, 10 % glycerol, 10 mM MgCl2, 0.5 mM EDTA and 5mM ATP
StainingType: NEGATIVE / Material: Uranyl Formate
GridModel: Maxtaform / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMax: 298.15 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Number grids imaged: 1 / Number real images: 1097 / Average exposure time: 0.4 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 102101
CTF correctionSoftware - Name: CTFFIND3 / Software - details: implemented in Appion
Details: EMAN1 was used for phase flipping whole micrograph before particle extraction.
Startup modelType of model: EMDB MAP
EMDB ID:

6254


Details: This map was low passed filtered to 60 Angstrom resolution.
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 78118
Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 23.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 14678
DetailsDifference of Gaussians (DoG)-based automated particle picker, implemented in Appion image processing software was used

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