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- EMDB-32276: 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 ED5_USP14 | |||||||||
![]() | Complete map of the human proteasome holoenzyme in state ED5_USP14, with the RP/CP low-pass filtered to 4.5/3.0 angstrom and sharpened by a B-factor of -30/0 | |||||||||
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Function / homology | ![]() negative regulation of ERAD pathway / Impaired BRCA2 translocation to the nucleus / Impaired BRCA2 binding to SEM1 (DSS1) / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
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Method | ![]() ![]() | |||||||||
![]() | 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 | ![]() | 899.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 70.5 KB 70.5 KB | Display Display | ![]() |
Images | ![]() | 72.3 KB | ||
Others | ![]() ![]() ![]() ![]() | 908.1 MB 875.2 MB 874.8 MB 903.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7w3bMC ![]() 7w37C ![]() 7w38C ![]() 7w39C ![]() 7w3aC ![]() 7w3cC ![]() 7w3fC ![]() 7w3gC ![]() 7w3hC ![]() 7w3iC ![]() 7w3jC ![]() 7w3kC ![]() 7w3mC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | Complete map of the human proteasome holoenzyme in state ED5_USP14, with the RP/CP low-pass filtered to 4.5/3.0 angstrom and sharpened by a B-factor of -30/0 | ||||||||||||||||||||
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: Map of the human proteasome in state ED5 USP14...
File | emd_32276_additional_1.map | ||||||||||||
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Annotation | Map of the human proteasome in state ED5_USP14 after CP-masked refinement, low-pass filtered to 3.0 angstrom with no sharpening B-factor applied | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unfiltered, unsharpened raw map of the human proteasome...
File | emd_32276_additional_2.map | ||||||||||||
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Annotation | Unfiltered, unsharpened raw map of the human proteasome in state ED5_USP14 after CP-masked refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unfiltered, unsharpened raw map of the human proteasome...
File | emd_32276_additional_3.map | ||||||||||||
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Annotation | Unfiltered, unsharpened raw map of the human proteasome in state ED5_USP14 after RP-masked refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Map of the human proteasome in state ED5 USP14...
File | emd_32276_additional_4.map | ||||||||||||
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Annotation | Map of the human proteasome in state ED5_USP14 after RP-masked refinement, low-pass filtered to 4.5 angstrom and sharpened by a B-factor of -30 | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : 26S proteasome
+Supramolecule #1: 26S proteasome
+Supramolecule #2: 26S proteasome
+Supramolecule #3: ED5_USP14
+Supramolecule #4: Substrate
+Macromolecule #1: 26S protease regulatory subunit 7
+Macromolecule #2: 26S protease regulatory subunit 4
+Macromolecule #3: Isoform 2 of 26S proteasome regulatory subunit 8
+Macromolecule #4: 26S protease regulatory subunit 6B
+Macromolecule #5: 26S proteasome regulatory subunit 10B
+Macromolecule #6: 26S protease regulatory subunit 6A
+Macromolecule #7: Proteasome subunit alpha type-6
+Macromolecule #8: Proteasome subunit alpha type-2
+Macromolecule #9: Proteasome subunit alpha type-4
+Macromolecule #10: Proteasome subunit alpha type-7
+Macromolecule #11: Proteasome subunit alpha type-5
+Macromolecule #12: Isoform Long of Proteasome subunit alpha type-1
+Macromolecule #13: Proteasome subunit alpha type-3
+Macromolecule #14: Proteasome subunit beta type-6
+Macromolecule #15: Proteasome subunit beta type-7
+Macromolecule #16: Proteasome subunit beta type-3
+Macromolecule #17: Proteasome subunit beta type-2
+Macromolecule #18: Proteasome subunit beta type-5
+Macromolecule #19: Proteasome subunit beta type-1
+Macromolecule #20: Proteasome subunit beta type-4
+Macromolecule #21: 26S proteasome non-ATPase regulatory subunit 1
+Macromolecule #22: 26S proteasome non-ATPase regulatory subunit 3
+Macromolecule #23: 26S proteasome non-ATPase regulatory subunit 12
+Macromolecule #24: 26S proteasome non-ATPase regulatory subunit 11
+Macromolecule #25: 26S proteasome non-ATPase regulatory subunit 6
+Macromolecule #26: 26S proteasome non-ATPase regulatory subunit 7
+Macromolecule #27: 26S proteasome non-ATPase regulatory subunit 13
+Macromolecule #28: 26S proteasome non-ATPase regulatory subunit 4
+Macromolecule #29: 26S proteasome non-ATPase regulatory subunit 14
+Macromolecule #30: 26S proteasome non-ATPase regulatory subunit 8
+Macromolecule #31: 26S proteasome non-ATPase regulatory subunit 2
+Macromolecule #32: Substrate
+Macromolecule #33: Ubiquitin carboxyl-terminal hydrolase 14
+Macromolecule #34: Ubiquitin
+Macromolecule #35: 26S proteasome complex subunit DSS1
+Macromolecule #36: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #37: ADENOSINE-5'-DIPHOSPHATE
+Macromolecule #38: ZINC ION
-Experimental details
-Structure determination
Method | ![]() |
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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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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2 |
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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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 35156 |