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- PDB-8qh5: CryoEM structure of UVSSA(VHS)-CSA-DDB1-DDA1 -

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Basic information

Entry
Database: PDB / ID: 8qh5
TitleCryoEM structure of UVSSA(VHS)-CSA-DDB1-DDA1
Components
  • DET1- and DDB1-associated protein 1
  • DNA damage-binding protein 1
  • DNA excision repair protein ERCC-8
  • UV-stimulated scaffold protein A
KeywordsLIGASE / Ubiquitin ligase / DNA repair
Function / homology
Function and homology information


RNA polymerase inhibitor activity / regulation of transcription-coupled nucleotide-excision repair / nucleotide-excision repair complex / response to auditory stimulus / single strand break repair / positive regulation by virus of viral protein levels in host cell / double-strand break repair via classical nonhomologous end joining / chromatin-protein adaptor activity / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei ...RNA polymerase inhibitor activity / regulation of transcription-coupled nucleotide-excision repair / nucleotide-excision repair complex / response to auditory stimulus / single strand break repair / positive regulation by virus of viral protein levels in host cell / double-strand break repair via classical nonhomologous end joining / chromatin-protein adaptor activity / 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 / RNA polymerase II complex binding / cullin family protein binding / viral release from host cell / site of DNA damage / response to X-ray / ectopic germ cell programmed cell death / positive regulation of viral genome replication / ubiquitin-like ligase-substrate adaptor activity / response to UV / proteasomal protein catabolic process / protein autoubiquitination / transcription-coupled nucleotide-excision repair / positive regulation of gluconeogenesis / positive regulation of DNA repair / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / regulation of circadian rhythm / DNA Damage Recognition in GG-NER / 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 / Wnt signaling pathway / nuclear matrix / protein polyubiquitination / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / site of double-strand break / chromosome / Neddylation / response to oxidative stress / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / perikaryon / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / chromosome, telomeric region / protein ubiquitination / DNA repair / apoptotic process / DNA damage response / negative regulation of apoptotic process / protein-containing complex binding / nucleolus / protein-containing complex / extracellular space / DNA binding / extracellular exosome / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
UV-stimulated scaffold protein A / : / : / Uncharacterized conserved protein (DUF2043) / UVSSA N-terminal domain / Zinc finger UVSSA-type profile. / DNA excision repair protein Rad28/ERCC8/Ckn1/ATCSA-1 / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase ...UV-stimulated scaffold protein A / : / : / Uncharacterized conserved protein (DUF2043) / UVSSA N-terminal domain / Zinc finger UVSSA-type profile. / DNA excision repair protein Rad28/ERCC8/Ckn1/ATCSA-1 / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / ENTH/VHS / 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 / WD domain, G-beta repeat / G-protein beta WD-40 repeat / 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
DNA excision repair protein ERCC-8 / DNA damage-binding protein 1 / UV-stimulated scaffold protein A / DET1- and DDB1-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLee, S.-H. / Sixma, T.K.
Funding support Netherlands, European Union, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)TOP 714.017.003 Netherlands
European Molecular Biology Organization (EMBO)ALTF 1159-2019European Union
Citation
Journal: Nat Commun / Year: 2024
Title: The small CRL4 ubiquitin ligase component DDA1 regulates transcription-coupled repair dynamics.
Authors: Diana A Llerena Schiffmacher / Shun-Hsiao Lee / Katarzyna W Kliza / Arjan F Theil / Masaki Akita / Angela Helfricht / Karel Bezstarosti / Camila Gonzalo-Hansen / Haico van Attikum / Matty ...Authors: Diana A Llerena Schiffmacher / Shun-Hsiao Lee / Katarzyna W Kliza / Arjan F Theil / Masaki Akita / Angela Helfricht / Karel Bezstarosti / Camila Gonzalo-Hansen / Haico van Attikum / Matty Verlaan-de Vries / Alfred C O Vertegaal / Jan H J Hoeijmakers / Jurgen A Marteijn / Hannes Lans / Jeroen A A Demmers / Michiel Vermeulen / Titia K Sixma / Tomoo Ogi / Wim Vermeulen / Alex Pines /
Abstract: Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional ...Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4). Although ubiquitination of several TC-NER proteins by CRL4 has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4 complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
#1: Journal: To Be Published
Title: CryoEM structure of UVSSA(VHS)-CSA-DDB1-DDA1
Authors: Lee, S.-H. / Sixma, T.K.
History
DepositionSep 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 27, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: UV-stimulated scaffold protein A
A: DNA excision repair protein ERCC-8
B: DNA damage-binding protein 1
C: DET1- and DDB1-associated protein 1


Theoretical massNumber of molelcules
Total (without water)274,6854
Polymers274,6854
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein UV-stimulated scaffold protein A


Mass: 82923.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The construct contains an N-terminal His tag. / Source: (gene. exp.) Homo sapiens (human) / Gene: UVSSA, KIAA1530 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q2YD98
#2: Protein DNA excision repair protein ERCC-8 / Cockayne syndrome WD repeat protein CSA


Mass: 45465.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The construct contains a Strep tag II at the C-terminus
Source: (gene. exp.) Homo sapiens (human) / Gene: ERCC8, CKN1, CSA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13216
#3: 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: 129298.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The construct contains an N-terminal His tag. / Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q16531
#4: Protein DET1- and DDB1-associated protein 1 / Placenta cross-immune reaction antigen 1 / PCIA-1


Mass: 16997.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The construct contains a TwinStrep tag and a Flag tag at the C-terminus.
Source: (gene. exp.) Homo sapiens (human) / Gene: DDA1, C19orf58, PCIA1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BW61
Has protein modificationN

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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: Ternary complex of UVSSA(VHS)-CSA-DDB1-DDA1 / Type: COMPLEX
Details: Ternary complex of ubiquitinated UVSSA-USP7-CSA-DDB1-DDA1. USP7 is invisible in the cryoEM map.
Entity ID: #2-#4, #1 / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMsodium chlorideNaCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was glutaraldehyde crosslinked
Specimen supportDetails: The grid was coated with graphene oxide. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Collected on Krios 1 at Netherlands Center for Electron Nanoscopy (NeCEN)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1382
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
4cryoSPARCCTF correction
5RELIONCTF correction
8UCSF Chimeramodel fitting
9PHENIXmodel fitting
10Cootmodel fitting
12PHENIXmodel refinement
13cryoSPARCinitial Euler assignment
14RELION3.1.3initial Euler assignment
15RELION3.1.3final Euler assignment
17RELION3.3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 723908
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 294142
Details: Combined from focused maps reconstructed with various particle numbers.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Additional densities are observed near several cysteine residues (A/Cys222, A/Cys260, A/Cys288, B/Cys363, B/Cys725, B/Cys1008). We expect these are oxidized products or crosslinking side products.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeDetailsInitial refinement model-IDPdb chain residue rangeSource nameType
17OO3

7oo3

P7OO3P1-396CSA11-396PDBexperimental model
27OO3

7oo3

T7OO3T1-1140DDB1(BPA/BPC)11-1140PDBexperimental model
36ZX9

6zx9

A6ZX9A393-709DDB1(BPB)2393-709PDBexperimental model
46UD7

6ud7

D6UD7D4-76DDA134-76PDBexperimental model
5Q2YD98A1-150UVSSA(VHS)4AlphaFoldin silico model

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