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

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Basic information

Entry
Database: PDB / ID: 5jm9
TitleStructure of S. cerevesiae mApe1 dodecamer
ComponentsVacuolar aminopeptidase 1
KeywordsHYDROLASE / dodecamer / aminopeptidase / vacuole / cvt
Function / homology
Function and homology information


aminopeptidase I / Cvt complex / cytoplasm to vacuole targeting by the Cvt pathway / fungal-type vacuole / metalloaminopeptidase activity / protein transport / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 24 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, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 15, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2016Group: Database references
Revision 1.2Jun 29, 2016Group: Database references
Revision 1.3Jul 13, 2016Group: Database references
Revision 1.4Aug 2, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_image_scans / em_software / Item: _em_3d_fitting.target_criteria / _em_software.name
Revision 1.5Aug 30, 2017Group: Data collection / Category: em_software / Item: _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Vacuolar aminopeptidase 1


Theoretical massNumber of molelcules
Total (without water)57,1621
Polymers57,1621
Non-polymers00
Water0
1
A: Vacuolar aminopeptidase 1
x 12


Theoretical massNumber of molelcules
Total (without water)685,94912
Polymers685,94912
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation11

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Components

#1: Protein Vacuolar aminopeptidase 1 / Aminopeptidase yscI / Leucine aminopeptidase IV / LAPIV / Lysosomal aminopeptidase III / ...Aminopeptidase yscI / Leucine aminopeptidase IV / LAPIV / Lysosomal aminopeptidase III / Polypeptidase / Vacuolar aminopeptidase I


Mass: 57162.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: APE1, API, LAP4, YSC1, YKL103C, YKL455 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): RIL / References: UniProt: P14904, aminopeptidase I

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mature Aminopeptidase-1 dodecamer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 RIL / Plasmid: pETM33
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO / Details: The sample was purified using a GraFix gradient.
EM stainingType: NEGATIVE / Material: Uranyl acetate

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM12
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 53000 X / Alignment procedure: BASIC
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingElectron dose: 40 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1EMAN2.01particle selection
7UCSF Chimeramodel fitting
9EMAN2.1initial Euler assignment
10SPIDERfinal Euler assignment
12SPIDER19.093D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 5481
3D reconstructionResolution: 24 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 5481 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 4R8F

4r8f

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