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- EMDB-12047: CryoEM Structure of the yeast peroxisomal membrane Pex14p/Pex17p ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12047
TitleCryoEM Structure of the yeast peroxisomal membrane Pex14p/Pex17p complex
Map dataCryoEM Structure of the Pex14p/Pex17p complex
Sample
  • Complex: Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1D1dH4-6 nanodiscs
    • Protein or peptide: Peroxisomal membrane protein Pex14p
    • Protein or peptide: Peroxisomal membrane protein Pex17p
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.2 Å
AuthorsLill P / Gatsogiannis C
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR1905 Germany
Max Planck Society Germany
German Research Foundation (DFG)278002225;403222702;390939984 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Towards the molecular architecture of the peroxisomal receptor docking complex.
Authors: Pascal Lill / Tobias Hansen / Daniel Wendscheck / Bjoern Udo Klink / Tomasz Jeziorek / Dimitrios Vismpas / Jonas Miehling / Julian Bender / Andreas Schummer / Friedel Drepper / Wolfgang ...Authors: Pascal Lill / Tobias Hansen / Daniel Wendscheck / Bjoern Udo Klink / Tomasz Jeziorek / Dimitrios Vismpas / Jonas Miehling / Julian Bender / Andreas Schummer / Friedel Drepper / Wolfgang Girzalsky / Bettina Warscheid / Ralf Erdmann / Christos Gatsogiannis /
Abstract: Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded ...Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.
History
DepositionDec 7, 2020-
Header (metadata) releaseDec 30, 2020-
Map releaseDec 30, 2020-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12047.png

Downloads & links

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Map

FileDownload / File: emd_12047.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM Structure of the Pex14p/Pex17p complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 300 pix.
= 327. Å		1.09 Å/pix.
x 300 pix.
= 327. Å		1.09 Å/pix.
x 300 pix.
= 327. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.0047121374 - 0.021273872
Average (Standard dev.)7.5998316e-05 (±0.0010614756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 327.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z327.000327.000327.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0050.0210.000

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Supplemental data

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Additional map: CryoEM structure of the rod domain of the...

Fileemd_12047_additional_1.map
AnnotationCryoEM structure of the rod domain of the Pex14p/Pex17p complex upon signal subtraction of the nanodisc density and subsequent refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1...

EntireName: Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1D1dH4-6 nanodiscs
Components
  • Complex: Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1D1dH4-6 nanodiscs
    • Protein or peptide: Peroxisomal membrane protein Pex14p
    • Protein or peptide: Peroxisomal membrane protein Pex17p

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Supramolecule #1: Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1...

SupramoleculeName: Peroxisomal Membrane Complex Pex14p/Pex17p reconstituted in cMSP1D1dH4-6 nanodiscs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Pex14p and Pex17p constitute the major components of the peroxisomal membrane docking complex. Pex14p and Pex17p form a 3:1 heterotetrameric complex.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 144 KDa

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Macromolecule #1: Peroxisomal membrane protein Pex14p

MacromoleculeName: Peroxisomal membrane protein Pex14p / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSDVVSKDRK ALFDSAVSFL KDESIKDAPL LKKIEFLKSK GLTEKEIEIA MKEPKKDGIV GDEVSKKIGS TENRASQDM YLYEAMPPTL PHRDWKDYFV MATATAGLLY GAYEVTRRYV IPNILPEAKS KLEGDKKEID D QFSKIDTV LNAIEAEQAE FRKKESETLK ...String:
MSDVVSKDRK ALFDSAVSFL KDESIKDAPL LKKIEFLKSK GLTEKEIEIA MKEPKKDGIV GDEVSKKIGS TENRASQDM YLYEAMPPTL PHRDWKDYFV MATATAGLLY GAYEVTRRYV IPNILPEAKS KLEGDKKEID D QFSKIDTV LNAIEAEQAE FRKKESETLK ELSDTIAELK QALVQTTRSR EKIEDEFRIV KLEVVNMQNT ID KFVSDND GMQELNNIQK EMESLKSLMN NRMESGNAQD NRLFSISPNG IPGIDTIPSA SEILAKMGMQ EES DKEKEN GSDANKDDNA VPAWKKAREQ TIDSNASIPE WQKNTAANEI SVPDWQNGQV EDSIP

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Macromolecule #2: Peroxisomal membrane protein Pex17p

MacromoleculeName: Peroxisomal membrane protein Pex17p / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTSINSFPRN IDWPSNIGIK KIEGTNPTVN AIKGLLYNGG SIYAFLYFVI AMFVEPTLQK QYQQRNDFSL FVLLRLRRI IAQLQKRLVM TPVSSLGFNE QNNFVERSTQ TSDDNIIRED NSHWAEMIYQ LQNMKQELQY F NRSSGQPS ESIDDFVFQI KMVTDQVELT ...String:
MTSINSFPRN IDWPSNIGIK KIEGTNPTVN AIKGLLYNGG SIYAFLYFVI AMFVEPTLQK QYQQRNDFSL FVLLRLRRI IAQLQKRLVM TPVSSLGFNE QNNFVERSTQ TSDDNIIRED NSHWAEMIYQ LQNMKQELQY F NRSSGQPS ESIDDFVFQI KMVTDQVELT DRSRAFSNKS RNIIQGIREI KGWFVNGQVP R

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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

Concentration0.15 mg/mL
BufferpH: 8 / Component:
ConcentrationFormula
50.0 mMTRIS
150.0 mMNaCl
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II
DetailsThe complex was reconstituted in circularMSP1D1D4-6 nanodiscs.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 3282 / Average exposure time: 15.0 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 361862
Startup modelType of model: INSILICO MODEL
In silico model: An initial model was computed from 2D class averages using VIPER of the SPIRE software package.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 82600
Initial angle assignmentType: OTHER / Software - Name: SPHIRE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final 3D classificationSoftware - Name: SPHIRE

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