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- EMDB-8167: Structure of S. cerevesiae mApe1 dodecamer -

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Basic information

Entry
Database: EMDB / ID: EMD-8167
TitleStructure of S. cerevesiae mApe1 dodecamer
Map dataNone
Sample
  • Complex: Mature Aminopeptidase-1 dodecamer
    • Protein or peptide: Vacuolar aminopeptidase 1
Keywordsdodecamer / aminopeptidase / vacuole / cvt / Hydrolase
Function / homology
Function and homology information


aminopeptidase I / Cvt complex / cytoplasm to vacuole targeting by the Cvt pathway / fungal-type vacuole / metalloaminopeptidase activity / proteolysis / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
Peptidase M18 / Peptidase M18, domain 2 / Aminopeptidase I zinc metalloprotease (M18)
Similarity search - Domain/homology
Vacuolar aminopeptidase 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsSachse C / Bertipaglia C
CitationJournal: EMBO Rep / Year: 2016
Title: Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.
Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias ...Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias Wilmanns / Marko Kaksonen / John Ag Briggs / Carsten Sachse /
Abstract: Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles ...Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.
History
DepositionApr 28, 2016-
Header (metadata) releaseMay 25, 2016-
Map releaseJun 15, 2016-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jm9
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8167.png

Downloads & links

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Map

FileDownload / File: emd_8167.map.gz / Format: CCP4 / Size: 7.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 125 pix.
= 237.5 Å		1.9 Å/pix.
x 125 pix.
= 237.5 Å		1.9 Å/pix.
x 125 pix.
= 237.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.0060240254 - 0.068955064
Average (Standard dev.)0.004071423 (±0.010020978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions125125125
Spacing125125125
CellA=B=C: 237.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z125125125
origin x/y/z0.0000.0000.000
length x/y/z237.500237.500237.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS125125125
D min/max/mean-0.0060.0690.004

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Supplemental data

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Sample components

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Entire : Mature Aminopeptidase-1 dodecamer

EntireName: Mature Aminopeptidase-1 dodecamer
Components
  • Complex: Mature Aminopeptidase-1 dodecamer
    • Protein or peptide: Vacuolar aminopeptidase 1

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Supramolecule #1: Mature Aminopeptidase-1 dodecamer

SupramoleculeName: Mature Aminopeptidase-1 dodecamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: Vacuolar aminopeptidase 1

MacromoleculeName: Vacuolar aminopeptidase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: aminopeptidase I
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 57.16243 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MEEQREILEQ LKKTLQMLTV EPSKNNQIAN EEKEKKENEN SWCILEHNYE DIAQEFIDFI YKNPTTYHVV SFFAELLDKH NFKYLSEKS NWQDSIGEDG GKFYTIRNGT NLSAFILGKN WRAEKGVGVI GSHVDALTVK LKPVSFKDTA EGYGRIAVAP Y GGTLNELW ...String:
MEEQREILEQ LKKTLQMLTV EPSKNNQIAN EEKEKKENEN SWCILEHNYE DIAQEFIDFI YKNPTTYHVV SFFAELLDKH NFKYLSEKS NWQDSIGEDG GKFYTIRNGT NLSAFILGKN WRAEKGVGVI GSHVDALTVK LKPVSFKDTA EGYGRIAVAP Y GGTLNELW LDRDLGIGGR LLYKKKGTNE IKSALVDSTP LPVCRIPSLA PHFGKPAEGP FDKEDQTIPV IGFPTPDEEG NE PPTDDEK KSPLFGKHCI HLLRYVAKLA GVEVSELIQM DLDLFDVQKG TIGGIGKHFL FAPRLDDRLC SFAAMIALIC YAK DVNTEE SDLFSTVTLY DNEEIGSLTR QGAKGGLLES VVERSSSAFT KKPVDLHTVW ANSIILSADV NHLYNPNFPE VYLK NHFPV PNVGITLSLD PNGHMATDVV GTALVEELAR RNGDKVQYFQ IKNNSRSGGT IGPSLASQTG ARTIDLGIAQ LSMHS IRAA TGSKDVGLGV KFFNGFFKHW RSVYDEFGEL

UniProtKB: Vacuolar aminopeptidase 1

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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: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES
150.0 mMSodium ChlorideNaCl
StainingType: NEGATIVE / Material: Uranyl acetate
DetailsThe sample was purified using a GraFix gradient.

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Electron microscopy

MicroscopeFEI/PHILIPS CM12
Image recordingFilm or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 53000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Particle selectionNumber selected: 5481
Startup modelType of model: OTHER
Details: 1. EMAN2: Gaussian blob 2. Manual assignment based on Euler angles related by tetrahedral symmetry. Both initial models converged to the same 24 Angstrom resolution structure after 20 cycles ...Details: 1. EMAN2: Gaussian blob 2. Manual assignment based on Euler angles related by tetrahedral symmetry. Both initial models converged to the same 24 Angstrom resolution structure after 20 cycles of iterative refinement with SPIDER.
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER (ver. 19.09) / Number images used: 5481
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER

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Atomic model buiding 1

Initial modelPDB ID:

4r8f


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-5jm9:
Structure of S. cerevesiae mApe1 dodecamer

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