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- EMDB-5923: Architecture and assembly of the archaeal Cdc48-20S proteasome -

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Basic information

Entry
Database: EMDB / ID: EMD-5923
TitleArchitecture and assembly of the archaeal Cdc48-20S proteasome
Map dataSingle-particle reconstruction of the archaeal Cdc48-20S proteasome
Sample
  • Sample: Archaeal proteasome Cdc48-20S
  • Protein or peptide: Cdc48
  • Protein or peptide: 20S alpha subunit
  • Protein or peptide: 20S beta subunit
KeywordsArchaeal proteasome / Cdc48 / architecture / single-particle EM
Function / homology
Function and homology information


macromolecule metabolic process / primary metabolic process / response to stimulus / : / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / ATP hydrolysis activity ...macromolecule metabolic process / primary metabolic process / response to stimulus / : / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Proteasome alpha subunit, archaeal / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily ...Proteasome alpha subunit, archaeal / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / Nucleophile aminohydrolases, N-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
VCP-like ATPase / Proteasome subunit alpha
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / negative staining / Resolution: 42.0 Å
AuthorsBarthelme D / Chen JZ / Grabenstatter J / Baker TA / Sauer RT
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Architecture and assembly of the archaeal Cdc48*20S proteasome.
Authors: Dominik Barthelme / James Z Chen / Jonathan Grabenstatter / Tania A Baker / Robert T Sauer /
Abstract: ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of ...ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48 ⋅ 20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.
History
DepositionMar 13, 2014-
Header (metadata) releaseApr 9, 2014-
Map releaseApr 9, 2014-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5923.gif

Downloads & links

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Map

FileDownload / File: emd_5923.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle reconstruction of the archaeal Cdc48-20S proteasome
Voxel sizeX=Y=Z: 7.05 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-4.16351271 - 10.672816279999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 451.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.057.057.05
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z451.200451.200451.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-4.16410.6730.000

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Supplemental data

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Sample components

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Entire : Archaeal proteasome Cdc48-20S

EntireName: Archaeal proteasome Cdc48-20S
Components
  • Sample: Archaeal proteasome Cdc48-20S
  • Protein or peptide: Cdc48
  • Protein or peptide: 20S alpha subunit
  • Protein or peptide: 20S beta subunit

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Supramolecule #1000: Archaeal proteasome Cdc48-20S

SupramoleculeName: Archaeal proteasome Cdc48-20S / type: sample / ID: 1000
Oligomeric state: One homo-hexamer of Cdc48 binds to one homo-heptamer of 20S
Number unique components: 3
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Cdc48

MacromoleculeName: Cdc48 / type: protein_or_peptide / ID: 1 / Name.synonym: p97/VCP/VAT / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728
Molecular weightTheoretical: 500 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET22b
SequenceUniProtKB: VCP-like ATPase

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Macromolecule #2: 20S alpha subunit

MacromoleculeName: 20S alpha subunit / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728
Molecular weightTheoretical: 360 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pRSET-A
SequenceUniProtKB: Proteasome subunit alpha

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Macromolecule #3: 20S beta subunit

MacromoleculeName: 20S beta subunit / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728
Molecular weightTheoretical: 360 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3)
SequenceUniProtKB: Proteasome subunit alpha

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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

Concentration0.023 mg/mL
BufferpH: 7.5 / Details: 50mM HEPES, 100mM KCl, 20mM MgCl2
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 15 seconds.
GridDetails: 400 mesh Cu-grid with thin carbon support, glow-discharged before specimen loading
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42857 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: JEOL
TemperatureMin: 290 K / Max: 300 K / Average: 295 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 30,000 times magnification
DetailsWeak beam illumination
DateSep 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 183 / Average electron dose: 20 e/Å2 / Details: Every image was 2x-binned before processing. / Od range: 1.4 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

Final two d classificationNumber classes: 2612
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.0 Å / Resolution method: OTHER / Software - Name: PARTICLE / Number images used: 4724
DetailsParticles were selected manually using the PARTICLE package.

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