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- EMDB-32288: Structure of USP14-bound human 26S proteasome in substrate-engage... -
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Open data
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Basic information
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Title | Structure of USP14-bound human 26S proteasome in substrate-engaged state ED4_USP14 with the USP14 density improved | |||||||||
![]() | Map of the human 26S proteasome in state ED4_USP14, with the RP/CP low-pass filtered to 4.1/2.8 angstrom and sharpened by a B-factor of -100/0, rescaled to the pixel size of 0.685 angstrom | |||||||||
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![]() | Proteasome / AAA-ATPase / Deubiquitinase / USP14 / HYDROLASE | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
![]() | Zhang S / Zou S / Yin D / Wu Z / Mao Y | |||||||||
Funding support | ![]()
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![]() | ![]() Title: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM. Authors: Shuwen Zhang / Shitao Zou / Deyao Yin / Lihong Zhao / Daniel Finley / Zhaolong Wu / Youdong Mao / ![]() ![]() Abstract: Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating ...Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 930.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.1 KB 15.1 KB | Display Display | ![]() |
Images | ![]() | 96.4 KB | ||
Filedesc metadata | ![]() | 4.1 KB | ||
Others | ![]() | 59.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7w37C ![]() 7w38C ![]() 7w39C ![]() 7w3aC ![]() 7w3bC ![]() 7w3cC ![]() 7w3fC ![]() 7w3gC ![]() 7w3hC ![]() 7w3iC ![]() 7w3jC ![]() 7w3kC ![]() 7w3mC C: citing same article ( |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | Map of the human 26S proteasome in state ED4_USP14, with the RP/CP low-pass filtered to 4.1/2.8 angstrom and sharpened by a B-factor of -100/0, rescaled to the pixel size of 0.685 angstrom | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.685 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Unfiltered, unsharpened map of the human 26S proteasome...
File | emd_32288_additional_1.map | ||||||||||||
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Annotation | Unfiltered, unsharpened map of the human 26S proteasome in state ED4_USP14 | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : 26S proteasome
Entire | Name: 26S proteasome |
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Components |
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-Supramolecule #1: 26S proteasome
Supramolecule | Name: 26S proteasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#33 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 2.5 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.4 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: EMDB MAP EMDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 54642 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |