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- EMDB-1712: Three-dimensional CryoEM Structure of a Membrane-Anchored AAA Protease -

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Basic information

Entry
Database: EMDB / ID: EMD-1712
TitleThree-dimensional CryoEM Structure of a Membrane-Anchored AAA Protease
Map dataIsosurface representation of m-AAA protease. The density of transmembrane and intermembrane domains is weaker than those of AAA and protease domains. In the publication, the map was split into two parts and contoured separately, 91 kDa and 440 kDa.
Sample
  • Sample: m-AAA Protease
  • Protein or peptide: Yta10
  • Protein or peptide: Yta12
KeywordsAAA protein / protease / ATPase / membrane protein / mitocondria
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsLee S / Augustin S / Tatsuta T / Gerdes F / Langer T / Tsai FTF
CitationJournal: J Biol Chem / Year: 2011
Title: Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.
Authors: Sukyeong Lee / Steffen Augustin / Takashi Tatsuta / Florian Gerdes / Thomas Langer / Francis T F Tsai /
Abstract: FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, ...FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.
History
SupersessionID: EMD-1543
DepositionApr 13, 2010-
Header (metadata) releaseApr 26, 2011-
Map releaseApr 26, 2011-
UpdateJan 23, 2013-
Current statusJan 23, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

em1712.png

Downloads & links

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Map

FileDownload / File: emd_1712.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIsosurface representation of m-AAA protease. The density of transmembrane and intermembrane domains is weaker than those of AAA and protease domains. In the publication, the map was split into two parts and contoured separately, 91 kDa and 440 kDa.
Voxel sizeX=Y=Z: 1.81 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.150788 - 2.10365
Average (Standard dev.)0.105891 (±0.331558)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 231.68 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.811.811.81
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z231.680231.680231.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S213
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.1512.1040.106

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Supplemental data

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Sample components

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Entire : m-AAA Protease

EntireName: m-AAA Protease
Components
  • Sample: m-AAA Protease
  • Protein or peptide: Yta10
  • Protein or peptide: Yta12

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Supramolecule #1000: m-AAA Protease

SupramoleculeName: m-AAA Protease / type: sample / ID: 1000 / Details: Sample was cross-linked / Oligomeric state: One hetero hexamer of Yta10 and Yta12 / Number unique components: 2
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa / Method: From the amino acid sequence

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Macromolecule #1: Yta10

MacromoleculeName: Yta10 / type: protein_or_peptide / ID: 1 / Name.synonym: Yta10 / Details: Sample was cross-linked with Gluteraldehyde / Number of copies: 3 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Tissue: mitochondria / Cell: Saccharomyces cerevisiae / Organelle: mitochondria / Location in cell: mitochondrial inner membrane
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #2: Yta12

MacromoleculeName: Yta12 / type: protein_or_peptide / ID: 2 / Name.synonym: Yta12 / Details: Sample was cross-linked with Gluteraldehyde / Number of copies: 3 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Tissue: mitochondria / Cell: Saccharomyces cerevisiae / Organelle: mitochondria / Location in cell: mitochondrial inner membrane
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration0.3 mg/mL
BufferpH: 7.2
Details: 50mM Tris, pH 7.2, 150 mM NaCl, 1% n-octyl-beta-D-glucopyranoside, 10mM MgCl2, 5mM ATP
GridDetails: 400 mesh cupper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 83 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 6.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 400,000 x magnification
DateJun 5, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 17 e/Å2

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Image processing

CTF correctionDetails: Each CCD image
Final two d classificationNumber classes: 490
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: OTHER / Software - Name: EMAN / Number images used: 37882

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