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- PDB-7okq: Cryo-EM Structure of the DDB1-DCAF1-CUL4A-RBX1 Complex -

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Basic information

Entry
Database: PDB / ID: 7okq
TitleCryo-EM Structure of the DDB1-DCAF1-CUL4A-RBX1 Complex
Components
  • Cullin-4A
  • DDB1- and CUL4-associated factor 1
  • DNA damage-binding protein 1
  • E3 ubiquitin-protein ligase RBX1
KeywordsLIGASE / ubiquitin / E3 / protein degradation
Function / homology
Function and homology information


histone H2AT120 kinase activity / cell competition in a multicellular organism / negative regulation of granulocyte differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / V(D)J recombination / positive regulation by virus of viral protein levels in host cell / regulation of DNA damage checkpoint / cellular response to chemical stress ...histone H2AT120 kinase activity / cell competition in a multicellular organism / negative regulation of granulocyte differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / V(D)J recombination / positive regulation by virus of viral protein levels in host cell / regulation of DNA damage checkpoint / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / spindle assembly involved in female meiosis / regulation of nucleotide-excision repair / epigenetic programming in the zygotic pronuclei / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / Cul4-RING E3 ubiquitin ligase complex / positive regulation of protein autoubiquitination / UV-damage excision repair / protein neddylation / NEDD8 ligase activity / biological process involved in interaction with symbiont / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / ubiquitin-ubiquitin ligase activity / regulation of mitotic cell cycle phase transition / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4B-RING E3 ubiquitin ligase complex / negative regulation of type I interferon production / ubiquitin ligase complex scaffold activity / Cul3-RING ubiquitin ligase complex / Prolactin receptor signaling / negative regulation of reproductive process / negative regulation of developmental process / protein monoubiquitination / viral release from host cell / cullin family protein binding / somatic stem cell population maintenance / hemopoiesis / ectopic germ cell programmed cell death / ubiquitin-like ligase-substrate adaptor activity / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of viral genome replication / proteasomal protein catabolic process / protein K48-linked ubiquitination / Nuclear events stimulated by ALK signaling in cancer / positive regulation of gluconeogenesis / positive regulation of TORC1 signaling / post-translational protein modification / intrinsic apoptotic signaling pathway / regulation of cellular response to insulin stimulus / Regulation of BACH1 activity / B cell differentiation / T cell activation / nuclear estrogen receptor binding / nucleotide-excision repair / Degradation of DVL / cellular response to amino acid stimulus / Recognition of DNA damage by PCNA-containing replication complex / Degradation of GLI1 by the proteasome / Negative regulation of NOTCH4 signaling / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / regulation of circadian rhythm / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / DNA Damage Recognition in GG-NER / RING-type E3 ubiquitin transferase / negative regulation of canonical Wnt signaling pathway / Degradation of beta-catenin by the destruction complex / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Dual Incision in GG-NER / Evasion by RSV of host interferon responses / NOTCH1 Intracellular Domain Regulates Transcription / Wnt signaling pathway / fibrillar center / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / Regulation of expression of SLITs and ROBOs / Formation of Incision Complex in GG-NER / Interleukin-1 signaling / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / Orc1 removal from chromatin / protein polyubiquitination / Regulation of RAS by GAPs / G1/S transition of mitotic cell cycle / positive regulation of protein catabolic process / Regulation of RUNX2 expression and activity / cellular response to UV / MAPK cascade / ubiquitin protein ligase activity
Similarity search - Function
VPRBP/DCAF1 family / Lissencephaly type-1-like homology motif / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin family signature. ...VPRBP/DCAF1 family / Lissencephaly type-1-like homology motif / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / : / CPSF A subunit region / Zinc finger RING-type profile. / Zinc finger, RING-type / Armadillo-like helical / Armadillo-type fold / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase RBX1 / Cullin-4A / DNA damage-binding protein 1 / DDB1- and CUL4-associated factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.4 Å
AuthorsMohamed, W.I. / Schenk, A.D. / Kempf, G. / Cavadini, S. / Thoma, N.H.
CitationJournal: EMBO J / Year: 2021
Title: The CRL4 cullin-RING ubiquitin ligase is activated following a switch in oligomerization state.
Authors: Weaam I Mohamed / Andreas D Schenk / Georg Kempf / Simone Cavadini / Anja Basters / Alessandro Potenza / Wassim Abdul Rahman / Julius Rabl / Kurt Reichermeier / Nicolas H Thomä /
Abstract: The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4- ...The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4 ligase. In the 8.4 Å cryo-EM map of CRL4 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
History
DepositionMay 18, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jul 10, 2024Group: Data collection / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA damage-binding protein 1
B: DDB1- and CUL4-associated factor 1
C: Cullin-4A
D: E3 ubiquitin-protein ligase RBX1
E: DNA damage-binding protein 1
F: DDB1- and CUL4-associated factor 1
G: Cullin-4A
H: E3 ubiquitin-protein ligase RBX1
I: DNA damage-binding protein 1
J: DDB1- and CUL4-associated factor 1
K: Cullin-4A
L: E3 ubiquitin-protein ligase RBX1
M: DNA damage-binding protein 1
N: DDB1- and CUL4-associated factor 1
O: Cullin-4A
P: E3 ubiquitin-protein ligase RBX1


Theoretical massNumber of molelcules
Total (without water)1,603,49616
Polymers1,603,49616
Non-polymers00
Water00
1
A: DNA damage-binding protein 1
B: DDB1- and CUL4-associated factor 1
C: Cullin-4A
D: E3 ubiquitin-protein ligase RBX1

E: DNA damage-binding protein 1
F: DDB1- and CUL4-associated factor 1
G: Cullin-4A
H: E3 ubiquitin-protein ligase RBX1

I: DNA damage-binding protein 1
J: DDB1- and CUL4-associated factor 1
K: Cullin-4A
L: E3 ubiquitin-protein ligase RBX1

M: DNA damage-binding protein 1
N: DDB1- and CUL4-associated factor 1
O: Cullin-4A
P: E3 ubiquitin-protein ligase RBX1


Theoretical massNumber of molelcules
Total (without water)1,603,49616
Polymers1,603,49616
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3

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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: 129394.898 Da / Num. of mol.: 4
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 1 / HIV-1 Vpr-binding protein / VprBP / Serine/threonine-protein kinase VPRBP / Vpr-interacting protein


Mass: 171279.094 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DCAF1, KIAA0800, RIP, VPRBP / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q9Y4B6, non-specific serine/threonine protein kinase
#3: Protein
Cullin-4A / CUL-4A


Mass: 86702.836 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL4A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13619
#4: Protein
E3 ubiquitin-protein ligase RBX1 / E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / ...E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / Rbx1 / Regulator of cullins 1 / ROC1


Mass: 13497.240 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P62877, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase

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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: CRL4(DCAF1) / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
7Cootmodel fitting
8UCSF Chimeramodel fitting
11RosettaEMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14000 / Symmetry type: POINT
Atomic model buildingB value: 596 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Real-space cross-correlation
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12HYE

2hye

12HYE1PDBexperimental model
25JK7

5jk7

15JK72PDBexperimental model

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