+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5923 | |||||||||
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Title | Architecture and assembly of the archaeal Cdc48-20S proteasome | |||||||||
Map data | Single-particle reconstruction of the archaeal Cdc48-20S proteasome | |||||||||
Sample |
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Keywords | Archaeal proteasome / Cdc48 / architecture / single-particle EM | |||||||||
Function / homology | Function and homology information retrograde protein transport, ER to cytosol / polyubiquitin modification-dependent protein binding / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / threonine-type endopeptidase activity / endopeptidase activity / ubiquitin-dependent protein catabolic process / ATP hydrolysis activity / ATP binding / cytosol Similarity search - Function | |||||||||
Biological species | Thermoplasma acidophilum (acidophilic) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 42.0 Å | |||||||||
Authors | Barthelme D / Chen JZ / Grabenstatter J / Baker TA / Sauer RT | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5923.map.gz | 956.4 KB | EMDB map data format | |
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Header (meta data) | emd-5923-v30.xml emd-5923.xml | 12.4 KB 12.4 KB | Display Display | EMDB header |
Images | 400_5923.gif 80_5923.gif | 37.2 KB 3.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5923 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5923 | HTTPS FTP |
-Validation report
Summary document | emd_5923_validation.pdf.gz | 78.3 KB | Display | EMDB validaton report |
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Full document | emd_5923_full_validation.pdf.gz | 77.4 KB | Display | |
Data in XML | emd_5923_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5923 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5923 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5923.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Single-particle reconstruction of the archaeal Cdc48-20S proteasome | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 7.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Archaeal proteasome Cdc48-20S
Entire | Name: Archaeal proteasome Cdc48-20S |
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Components |
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-Supramolecule #1000: Archaeal proteasome Cdc48-20S
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 1.2 MDa |
-Macromolecule #1: Cdc48
Macromolecule | Name: Cdc48 / type: protein_or_peptide / ID: 1 / Name.synonym: p97/VCP/VAT / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728 |
Molecular weight | Theoretical: 500 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET22b |
Sequence | UniProtKB: VCP-like ATPase |
-Macromolecule #2: 20S alpha subunit
Macromolecule | Name: 20S alpha subunit / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes |
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Source (natural) | Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728 |
Molecular weight | Theoretical: 360 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pRSET-A |
Sequence | UniProtKB: Proteasome subunit alpha |
-Macromolecule #3: 20S beta subunit
Macromolecule | Name: 20S beta subunit / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes |
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Source (natural) | Organism: Thermoplasma acidophilum (acidophilic) / Strain: DSM 1728 |
Molecular weight | Theoretical: 360 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) |
Sequence | UniProtKB: Proteasome subunit alpha |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.023 mg/mL |
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Buffer | pH: 7.5 / Details: 50mM HEPES, 100mM KCl, 20mM MgCl2 |
Staining | Type: NEGATIVE Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 15 seconds. |
Grid | Details: 400 mesh Cu-grid with thin carbon support, glow-discharged before specimen loading |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | JEOL 2100F |
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Temperature | Min: 290 K / Max: 300 K / Average: 295 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 30,000 times magnification |
Details | Weak beam illumination |
Date | Sep 1, 2013 |
Image recording | Category: 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 min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 42857 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 30000 |
Sample stage | Specimen holder model: JEOL |
-Image processing
Details | Particles were selected manually using the PARTICLE package. |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.0 Å / Resolution method: OTHER / Software - Name: PARTICLE / Number images used: 4724 |
Final two d classification | Number classes: 2612 |