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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


cell competition in a multicellular organism / histone H2AT120 kinase activity / V(D)J recombination / negative regulation of beige fat cell differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / negative regulation of mitophagy / cullin-RING ubiquitin ligase complex / regulation of xenophagy / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling ...cell competition in a multicellular organism / histone H2AT120 kinase activity / V(D)J recombination / negative regulation of beige fat cell differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / negative regulation of mitophagy / cullin-RING ubiquitin ligase complex / regulation of xenophagy / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / regulation of cell cycle process / neural crest cell differentiation / RNA polymerase II transcription initiation surveillance / positive regulation by virus of viral protein levels in host cell / positive regulation of protein autoubiquitination / protein neddylation / spindle assembly involved in female meiosis / regulation of BMP signaling pathway / NEDD8 ligase activity / epigenetic programming in the zygotic pronuclei / regulation of mitophagy / negative regulation of response to oxidative stress / regulation of centrosome duplication / UV-damage excision repair / protein K27-linked ubiquitination / VCB complex / Cul5-RING ubiquitin ligase complex / regulation of TOR signaling / ubiquitin-ubiquitin ligase activity / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Cul2-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / biological process involved in interaction with symbiont / negative regulation of DNA-templated DNA replication / regulation of mitotic cytokinesis / Cul3-RING ubiquitin ligase complex / regulation of DNA damage checkpoint / regulation of mitotic cell cycle phase transition / negative regulation of type I interferon production / regulation of miRNA-mediated gene silencing / regulation of natural killer cell activation / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / WD40-repeat domain binding / Prolactin receptor signaling / regulation of cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / regulation of stem cell population maintenance / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of adipose tissue development / regulation of cellular response to stress / limb development / viral release from host cell / protein monoubiquitination / cullin family protein binding / centrosome duplication / regulation of DNA-templated DNA replication initiation / positive regulation of viral genome replication / positive regulation of gluconeogenesis / cilium assembly / B cell differentiation / ubiquitin-like ligase-substrate adaptor activity / intrinsic apoptotic signaling pathway / ribosome-associated ubiquitin-dependent protein catabolic process / signal transduction in response to DNA damage / negative regulation of insulin receptor signaling pathway / Nuclear events stimulated by ALK signaling in cancer / protein K48-linked ubiquitination / regulation of cellular response to insulin stimulus / positive regulation of TORC1 signaling / in utero embryonic development / transcription-coupled nucleotide-excision repair / post-translational protein modification / cellular response to amino acid stimulus / regulation of embryonic development / replication fork processing / nuclear estrogen receptor binding / negative regulation of canonical NF-kappaB signal transduction / regulation of mitotic cell cycle / Regulation of BACH1 activity / site of DNA damage / T cell activation / G1/S transition of mitotic cell cycle / proteasomal protein catabolic process / negative regulation of canonical Wnt signaling pathway / epigenetic regulation of gene expression / Degradation of DVL / Degradation of CRY and PER proteins / nucleotide-excision repair / Degradation of GLI1 by the proteasome / Recognition of DNA damage by PCNA-containing replication complex / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Negative regulation of NOTCH4 signaling / regulation of autophagy / fibrillar center / RING-type E3 ubiquitin transferase / Hedgehog 'on' state
Similarity search - Function
VPRBP/DCAF1 family / DCAF1 helical domain / DCAF1 fourth domain / Mahjong helical domain / DCAF1 N-terminal domain / Lissencephaly type-1-like homology motif / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Zinc finger, RING-H2-type / RING-H2 zinc finger domain ...VPRBP/DCAF1 family / DCAF1 helical domain / DCAF1 fourth domain / Mahjong helical domain / DCAF1 N-terminal domain / Lissencephaly type-1-like homology motif / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin, N-terminal / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin alpha solenoid domain / Cullin / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / : / 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 / 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 / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing 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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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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