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- EMDB-6496: Electron microscopy of apo yeast Rad4 (XPC homolog) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-6496
TitleElectron microscopy of apo yeast Rad4 (XPC homolog) complex
Map dataReconstruction of apo yeast Rad4 (XPC homolog) complex
Sample
  • Sample: Apo yeast Rad4 complex
  • Protein or peptide: RAD4
  • Protein or peptide: RAD23
  • Protein or peptide: RAD33
Keywordstranscription / DNA repair / stem cells
Function / homology
Function and homology information


PNGase complex / nucleotide-excision repair factor 2 complex / single-strand break-containing DNA binding / ubiquitin-dependent glycoprotein ERAD pathway / XPC complex / nucleotide-excision repair, DNA damage recognition / SUMOylation of DNA damage response and repair proteins / protein deglycosylation / proteasome binding / DNA topological change ...PNGase complex / nucleotide-excision repair factor 2 complex / single-strand break-containing DNA binding / ubiquitin-dependent glycoprotein ERAD pathway / XPC complex / nucleotide-excision repair, DNA damage recognition / SUMOylation of DNA damage response and repair proteins / protein deglycosylation / proteasome binding / DNA topological change / polyubiquitin modification-dependent protein binding / mismatch repair / : / ubiquitin binding / nucleotide-excision repair / protein-macromolecule adaptor activity / single-stranded DNA binding / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / negative regulation of transcription by RNA polymerase II / mitochondrion / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Rad33 / Rad33 / Rad33 / DNA repair protein Rad4 / DNA repair protein Rad4 / DNA repair protein Rad4, subgroup / Rad4/PNGase transglutaminase-like fold / Rad4 beta-hairpin domain 1 / Rad4 beta-hairpin domain 2 / Rad4 beta-hairpin domain 3 ...Rad33 / Rad33 / Rad33 / DNA repair protein Rad4 / DNA repair protein Rad4 / DNA repair protein Rad4, subgroup / Rad4/PNGase transglutaminase-like fold / Rad4 beta-hairpin domain 1 / Rad4 beta-hairpin domain 2 / Rad4 beta-hairpin domain 3 / Rad4, beta-hairpin domain 3 superfamily / Rad4 beta-hairpin domain 1 / Rad4 beta-hairpin domain 3 / Rad4 beta-hairpin domain 1 / Rad4 beta-hairpin domain 2 / Rad4 beta-hairpin domain 3 / Rad4 transglutaminase-like domain / UV excision repair protein Rad23 / UV excision repair protein Rad23 / XPC-binding domain / XPC-binding domain superfamily / XPC-binding domain / Heat shock chaperonin-binding / Heat shock chaperonin-binding motif. / Transglutaminase-like superfamily / UBA/TS-N domain / Ubiquitin associated domain / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily / Papain-like cysteine peptidase superfamily / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
DNA repair protein RAD4 / UV excision repair protein RAD23 / DNA repair protein RAD33
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsZhang ET / He Y / Grob P / Fong YW / Nogales E / Tjian R
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Architecture of the human XPC DNA repair and stem cell coactivator complex.
Authors: Elisa T Zhang / Yuan He / Patricia Grob / Yick W Fong / Eva Nogales / Robert Tjian /
Abstract: The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the ...The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.
History
DepositionNov 1, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseNov 18, 2015-
UpdateDec 16, 2015-
Current statusDec 16, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.35
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.35
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6496.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of apo yeast Rad4 (XPC homolog) complex
Voxel sizeX=Y=Z: 3.01 Å
Density
Contour LevelBy AUTHOR: 5.35 / Movie #1: 5.35
Minimum - Maximum-5.30278921 - 16.28396416
Average (Standard dev.)0.0 (±0.99999976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.013.013.01
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z385.280385.280385.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-189
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-5.30316.2840.000

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Supplemental data

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Sample components

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Entire : Apo yeast Rad4 complex

EntireName: Apo yeast Rad4 complex
Components
  • Sample: Apo yeast Rad4 complex
  • Protein or peptide: RAD4
  • Protein or peptide: RAD23
  • Protein or peptide: RAD33

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Supramolecule #1000: Apo yeast Rad4 complex

SupramoleculeName: Apo yeast Rad4 complex / type: sample / ID: 1000
Details: Thawed from -80 degrees Celsius and placed on ice immediately prior to grid preparation.
Oligomeric state: heterotrimer / Number unique components: 3
Molecular weightExperimental: 175 KDa / Theoretical: 175 KDa / Method: SDS-PAGE

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Macromolecule #1: RAD4

MacromoleculeName: RAD4 / type: protein_or_peptide / ID: 1 / Name.synonym: DNA repair protein RAD4 / Details: contains N-terminal 6xHis and TEV cleavage site / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: nucleus, cytoplasm
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
SequenceUniProtKB: DNA repair protein RAD4 / GO: nucleotide-excision repair factor 2 complex / InterPro: DNA repair protein Rad4

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Macromolecule #2: RAD23

MacromoleculeName: RAD23 / type: protein_or_peptide / ID: 2 / Name.synonym: UV excision repair protein RAD23 / Details: contains N-terminal 1xFLAG tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: nucleus, cytoplasm
Molecular weightExperimental: 55 KDa / Theoretical: 55 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
SequenceUniProtKB: UV excision repair protein RAD23 / GO: nucleotide-excision repair factor 2 complex / InterPro: UV excision repair protein Rad23

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Macromolecule #3: RAD33

MacromoleculeName: RAD33 / type: protein_or_peptide / ID: 3 / Name.synonym: DNA repair protein RAD33 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: nucleus
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
SequenceUniProtKB: DNA repair protein RAD33 / InterPro: Rad33

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.6
Details: 300 mM KCl, 50 mM HEPES, 0.1% NP-40 alternative, 10% glycerol, 0.1 mM EDTA, 1 mM MgCl2, 1 mM TCEP, 1 mM DTT
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 2 successive droplets of buffer G (300 mM KCl, 25 mM HEPES ph 7.6, 3% w/v trehalose, 0.01% NP-40 alternative, 1 mM TCEP, 1 mM DTT, 0.1 mM EDTA, 1 ...Details: Grids with adsorbed protein were floated on 2 successive droplets of buffer G (300 mM KCl, 25 mM HEPES ph 7.6, 3% w/v trehalose, 0.01% NP-40 alternative, 1 mM TCEP, 1 mM DTT, 0.1 mM EDTA, 1 mM MgCl2) and subsequently floated on 4 successive 1% w/v uranyl droplets for 10 seconds each.
GridDetails: 400 mesh copper grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.51 µm / Nominal defocus min: 0.49 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 80,000 times and 280,000 times magnification.
DateAug 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 486 / Average electron dose: 30 e/Å2 / Bits/pixel: 32
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 12879
DetailsParticles were selected by DoGPicker in the Appion pipeline.

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