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- EMDB-6265: Three-dimensional reconstruction of yeast Ape1 -

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Basic information

Entry
Database: EMDB / ID: EMD-6265
TitleThree-dimensional reconstruction of yeast Ape1
Map dataReconstruction of yeast Ape1
Sample
  • Sample: aminopeptidase 1 of yeast
  • Protein or peptide: Aminopeptidase 1
KeywordsAutophagic vesicle formation
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 19.0 Å
AuthorsSu MY / Su SC / Chang YC / Chang CI
CitationJournal: Autophagy / Year: 2015
Title: Structure of yeast Ape1 and its role in autophagic vesicle formation.
Authors: Ming-Yuan Su / Wen-Hsin Peng / Meng-Ru Ho / Shih-Chieh Su / Yuan-Chih Chang / Guang-Chao Chen / Chung-I Chang /
Abstract: In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the ...In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.
History
DepositionFeb 8, 2015-
Header (metadata) releaseMar 18, 2015-
Map releaseMar 18, 2015-
UpdateMar 25, 2015-
Current statusMar 25, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6265.jpg

Downloads & links

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Map

FileDownload / File: emd_6265.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast Ape1
Voxel sizeX=Y=Z: 1.65 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.0002
Minimum - Maximum-0.00171373 - 0.00231679
Average (Standard dev.)-0.0000931 (±0.00038824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 184.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.651.651.65
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z184.800184.800184.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-0.0020.002-0.000

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Supplemental data

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Sample components

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Entire : aminopeptidase 1 of yeast

EntireName: aminopeptidase 1 of yeast
Components
  • Sample: aminopeptidase 1 of yeast
  • Protein or peptide: Aminopeptidase 1

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Supramolecule #1000: aminopeptidase 1 of yeast

SupramoleculeName: aminopeptidase 1 of yeast / type: sample / ID: 1000 / Oligomeric state: One homododecamer of Ape1 / Number unique components: 1
Molecular weightExperimental: 720 KDa / Theoretical: 720 KDa / Method: Sedimentation

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Macromolecule #1: Aminopeptidase 1

MacromoleculeName: Aminopeptidase 1 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 720 KDa / Theoretical: 720 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 60 seconds.
GridDetails: 400 mesh gold grid with thin carbon support
VitrificationCryogen name: NITROGEN / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal magnification: 62000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJan 1, 2015
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

CTF correctionDetails: Each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 29586

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