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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


regulation of transcription-coupled nucleotide-excision repair / nucleotide-excision repair complex / single strand break repair / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / double-strand break repair via classical nonhomologous end joining / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont ...regulation of transcription-coupled nucleotide-excision repair / nucleotide-excision repair complex / single strand break repair / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / double-strand break repair via classical nonhomologous end joining / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / WD40-repeat domain binding / ubiquitin ligase complex scaffold activity / Cul4B-RING E3 ubiquitin ligase complex / negative regulation of reproductive process / negative regulation of developmental process / RNA polymerase II complex binding / cullin family protein binding / viral release from host cell / response to X-ray / ectopic germ cell programmed cell death / proteasomal protein catabolic process / protein autoubiquitination / transcription-coupled nucleotide-excision repair / positive regulation of viral genome replication / response to UV / positive regulation of gluconeogenesis / positive regulation of DNA repair / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / DNA Damage Recognition in GG-NER / regulation of circadian rhythm / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Dual Incision in GG-NER / Wnt signaling pathway / Formation of Incision Complex in GG-NER / nuclear matrix / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / protein polyubiquitination / cellular response to UV / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / rhythmic process / chromosome / Neddylation / protein-macromolecule adaptor activity / site of double-strand break / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / response to oxidative stress / damaged DNA binding / chromosome, telomeric region / protein ubiquitination / DNA repair / DNA damage response / protein-containing complex binding / apoptotic process / nucleolus / negative regulation of apoptotic process / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
UV-stimulated scaffold protein A / : / : / Uncharacterized conserved protein (DUF2043) / UVSSA N-terminal domain / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / DNA excision repair protein Rad28/ERCC8/Ckn1/ATCSA-1 / Det1 complexing ubiquitin ligase / ENTH/VHS ...UV-stimulated scaffold protein A / : / : / Uncharacterized conserved protein (DUF2043) / UVSSA N-terminal domain / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / DNA excision repair protein Rad28/ERCC8/Ckn1/ATCSA-1 / Det1 complexing ubiquitin ligase / ENTH/VHS / : / 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 / 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. / WD domain, G-beta repeat / 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
CitationJournal: 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

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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

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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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