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- PDB-8ajo: Negative-stain electron microscopy structure of DDB1-DCAF12-CCT5 -

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Basic information

Entry
Database: PDB / ID: 8ajo
TitleNegative-stain electron microscopy structure of DDB1-DCAF12-CCT5
Components
  • DDB1- and CUL4-associated factor 12
  • DNA damage-binding protein 1
  • T-complex protein 1 subunit epsilon
KeywordsLIGASE / chaperone / E3 / ubiquitin / DDB1 / DCAF12
Function / homology
Function and homology information


positive regulation of protein localization to Cajal body / positive regulation of telomerase RNA localization to Cajal body / chaperonin-containing T-complex / : / BBSome-mediated cargo-targeting to cilium / Formation of tubulin folding intermediates by CCT/TriC / Folding of actin by CCT/TriC / binding of sperm to zona pellucida / positive regulation by virus of viral protein levels in host cell / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway ...positive regulation of protein localization to Cajal body / positive regulation of telomerase RNA localization to Cajal body / chaperonin-containing T-complex / : / BBSome-mediated cargo-targeting to cilium / Formation of tubulin folding intermediates by CCT/TriC / Folding of actin by CCT/TriC / binding of sperm to zona pellucida / positive regulation by virus of viral protein levels in host cell / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Prefoldin mediated transfer of substrate to CCT/TriC / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of reproductive process / negative regulation of developmental process / Association of TriC/CCT with target proteins during biosynthesis / cullin family protein binding / viral release from host cell / ectopic germ cell programmed cell death / beta-tubulin binding / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / positive regulation of viral genome replication / ubiquitin-like ligase-substrate adaptor activity / proteasomal protein catabolic process / : / positive regulation of telomere maintenance via telomerase / positive regulation of gluconeogenesis / protein folding chaperone / T cell activation / Recognition of DNA damage by PCNA-containing replication complex / mRNA 3'-UTR binding / DNA Damage Recognition in GG-NER / nucleotide-excision repair / ATP-dependent protein folding chaperone / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Formation of Incision Complex in GG-NER / regulation of circadian rhythm / mRNA 5'-UTR binding / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / response to virus / Wnt signaling pathway / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / unfolded protein binding / site of double-strand break / G-protein beta-subunit binding / protein folding / Neddylation / cell body / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / damaged DNA binding / proteasome-mediated ubiquitin-dependent protein catabolic process / microtubule / chromosome, telomeric region / protein ubiquitination / protein stabilization / regulation of autophagy / DNA repair / apoptotic process / centrosome / DNA damage response / negative regulation of apoptotic process / protein-containing complex binding / nucleolus / protein-containing complex / ATP hydrolysis activity / extracellular space / DNA binding / extracellular exosome / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / : / DDB1- and CUL4-associated factor 12 beta propeller / T-complex protein 1, epsilon subunit / : / Chaperonins TCP-1 signature 1. / : / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. ...: / : / DDB1- and CUL4-associated factor 12 beta propeller / T-complex protein 1, epsilon subunit / : / Chaperonins TCP-1 signature 1. / : / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. / Chaperone tailless complex polypeptide 1 (TCP-1) / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
T-complex protein 1 subunit epsilon / DNA damage-binding protein 1 / DDB1- and CUL4-associated factor 12
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 30.6 Å
AuthorsPla-Prats, C. / Cavadini, S. / Kempf, G. / Thoma, N.H.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: EMBO J / Year: 2023
Title: Recognition of the CCT5 di-Glu degron by CRL4 is dependent on TRiC assembly.
Authors: Carlos Pla-Prats / Simone Cavadini / Georg Kempf / Nicolas H Thomä /
Abstract: Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4 ) E3 ligase preferentially ...Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the β-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4 E3 ligase in overseeing the assembly of a key cellular complex.
History
DepositionJul 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA damage-binding protein 1
B: DDB1- and CUL4-associated factor 12
C: T-complex protein 1 subunit epsilon


Theoretical massNumber of molelcules
Total (without water)245,5943
Polymers245,5943
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3130 Å2
ΔGint-31 kcal/mol
Surface area99590 Å2

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Components

#1: Protein DNA damage-binding protein 1 / DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / ...DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / HBV X-associated protein 1 / XAP-1 / UV-damaged DNA-binding factor / UV-damaged DNA-binding protein 1 / UV-DDB 1 / XPE-binding factor / XPE-BF / Xeroderma pigmentosum group E-complementing protein / XPCe


Mass: 129784.258 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16531
#2: Protein DDB1- and CUL4-associated factor 12 / Centrosome-related protein TCC52 / Testis cancer centrosome-related protein / WD repeat-containing protein 40A


Mass: 53373.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DCAF12, KIAA1892, TCC52, WDR40A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5T6F0
#3: Protein T-complex protein 1 subunit epsilon / TCP-1-epsilon / CCT-epsilon


Mass: 62436.809 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCT5, CCTE, KIAA0098 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P48643

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of DDB1-DCAF12-CCT5 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: Uranyl acetate

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI SPIRIT
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 200 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 30.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2923 / Symmetry type: POINT

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