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
Deposition
Mar 13, 2014
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Header (metadata) release
Apr 9, 2014
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Map release
Apr 9, 2014
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Update
May 14, 2014
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Current status
May 14, 2014
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: Archaeal proteasome Cdc48-dN-20S / type: sample / ID: 1000 Oligomeric state: One homo-hexamer of Cdc48-dN binds to one homo-heptamer of 20S Number unique components: 3
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
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Electron microscopy
Microscope
JEOL 2100F
Temperature
Min: 290 K / Max: 300 K / Average: 295 K
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected at 30,000 times magnification
Date
Oct 1, 2013
Image recording
Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 101 / Average electron dose: 20 e/Å2 / Details: Every image was 2x-binned before processing. / Bits/pixel: 8
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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