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- PDB-7k01: Structure of TFIIH in TFIIH/Rad4-Rad23-Rad33 DNA opening complex -

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Basic information

Entry
Database: PDB / ID: 7k01
TitleStructure of TFIIH in TFIIH/Rad4-Rad23-Rad33 DNA opening complex
Components
  • (DNA repair helicase ...) x 2
  • (General transcription and DNA repair factor IIH subunit ...) x 4
  • RNA polymerase II transcription factor B subunit 5
KeywordsNUCLEAR PROTEIN/Transferase / TFIIH / Rad4 / Rad4/23 / XPC / NER / Nucleotide Excision Repair / GG-NER / NUCLEAR PROTEIN / NUCLEAR PROTEIN-Transferase complex
Function / homology
Function and homology information


regulation of mitotic recombination / RNA polymerase II promoter clearance / phosphatidylinositol-5-phosphate binding / positive regulation of mitotic recombination / nucleotide-excision repair factor 3 complex / DNA translocase activity / nucleotide-excision repair, preincision complex assembly / transcription open complex formation at RNA polymerase II promoter / transcriptional start site selection at RNA polymerase II promoter / DNA 5'-3' helicase ...regulation of mitotic recombination / RNA polymerase II promoter clearance / phosphatidylinositol-5-phosphate binding / positive regulation of mitotic recombination / nucleotide-excision repair factor 3 complex / DNA translocase activity / nucleotide-excision repair, preincision complex assembly / transcription open complex formation at RNA polymerase II promoter / transcriptional start site selection at RNA polymerase II promoter / DNA 5'-3' helicase / phosphatidylinositol-3-phosphate binding / transcription factor TFIIH holo complex / transcription factor TFIIH core complex / DNA 3'-5' helicase / transcription preinitiation complex / poly(A)+ mRNA export from nucleus / DNA duplex unwinding / 3'-5' DNA helicase activity / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase II Pre-transcription Events / Formation of TC-NER Pre-Incision Complex / RNA Polymerase I Promoter Escape / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription by RNA polymerase I / ATPase activator activity / Dual incision in TC-NER / ATP-dependent activity, acting on DNA / DNA helicase activity / isomerase activity / nucleotide-excision repair / transcription initiation at RNA polymerase II promoter / ubiquitin protein ligase activity / 4 iron, 4 sulfur cluster binding / 5'-3' DNA helicase activity / double-stranded DNA binding / transcription by RNA polymerase II / damaged DNA binding / DNA repair / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / nucleus / metal ion binding / cytosol
Similarity search - Function
Bacterial type XPD DNA helicase, FeS cluster domain / TFIIH subunit Tfb4/GTF2H3 / Transcription factor Tfb4 / TFIIH C1-like domain / Ssl1-like / TFIIH subunit Ssl1/p44 / Ssl1-like / TFIIH C1-like domain / TFIIH C1-like domain / TFIIH p62 subunit, N-terminal ...Bacterial type XPD DNA helicase, FeS cluster domain / TFIIH subunit Tfb4/GTF2H3 / Transcription factor Tfb4 / TFIIH C1-like domain / Ssl1-like / TFIIH subunit Ssl1/p44 / Ssl1-like / TFIIH C1-like domain / TFIIH C1-like domain / TFIIH p62 subunit, N-terminal / TFIIH subunit Tfb1/GTF2H1 / TFIIH p62 subunit, N-terminal domain / BSD domain / BSD domain superfamily / BSD domain / BSD domain profile. / domain in transcription factors and synapse-associated proteins / RAD3/XPD family / Helicase XPB/Ssl2 / ERCC3/RAD25/XPB helicase, C-terminal domain / Helicase XPB/Ssl2, N-terminal domain / Helicase conserved C-terminal domain / ERCC3/RAD25/XPB C-terminal helicase / Helical and beta-bridge domain / Helical and beta-bridge domain / Transcription factor TFIIH subunit p52/Tfb2 / Transcription factor Tfb2, C-terminal domain / Transcription factor Tfb2 / Transcription factor Tfb2 (p52) C-terminal domain / TFIIH subunit TTDA/Tfb5 / TFB5-like superfamily / Transcription factor TFIIH complex subunit Tfb5 / Transcription factor TFIIH complex subunit Tfb5 / ATP-dependent helicase Rad3/Chl1-like / : / Helicase-like, DEXD box c2 type / DEAD2 / DEAD_2 / DEXDc2 / Helicase superfamily 1/2, DinG/Rad3-like / HELICc2 / ATP-dependent helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain, DinG/Rad3-type / Helicase C-terminal domain / Superfamilies 1 and 2 helicase ATP-binding type-2 domain profile. / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / C1-like domain superfamily / von Willebrand factor (vWF) type A domain / VWFA domain profile. / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / Zinc finger C2H2-type / helicase superfamily c-terminal domain / PH-like domain superfamily / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Zinc finger, RING/FYVE/PHD-type / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / General transcription and DNA repair factor IIH helicase subunit XPD/RAD3 / General transcription and DNA repair factor IIH subunit TFB1 / General transcription and DNA repair factor IIH helicase/translocase subunit XPB/SSL2 / General transcription and DNA repair factor IIH subunit TFB2 / General transcription and DNA repair factor IIH subunit SSL1 / General transcription and DNA repair factor IIH subunit TFB4 / General transcription and DNA repair factor IIH subunit TFB5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
Authorsvan Eeuwen, T. / Min, J.H. / Murakami, K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM123233 United States
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in nucleotide excision repair.
Authors: Trevor van Eeuwen / Yoonjung Shim / Hee Jong Kim / Tingting Zhao / Shrabani Basu / Benjamin A Garcia / Craig D Kaplan / Jung-Hyun Min / Kenji Murakami /
Abstract: The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor ...The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.
History
DepositionSep 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
1: General transcription and DNA repair factor IIH subunit TFB1
4: General transcription and DNA repair factor IIH subunit TFB4
7: DNA repair helicase RAD25
5: RNA polymerase II transcription factor B subunit 5
2: General transcription and DNA repair factor IIH subunit TFB2
0: DNA repair helicase RAD3
6: General transcription and DNA repair factor IIH subunit SSL1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,75513
Polymers415,0767
Non-polymers6796
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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General transcription and DNA repair factor IIH subunit ... , 4 types, 4 molecules 1426

#1: Protein General transcription and DNA repair factor IIH subunit TFB1 / TFIIH subunit TFB1 / RNA polymerase II transcription factor B 73 kDa subunit / RNA polymerase II ...TFIIH subunit TFB1 / RNA polymerase II transcription factor B 73 kDa subunit / RNA polymerase II transcription factor B p73 subunit / RNA polymerase II transcription factor B subunit 1


Mass: 72993.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32776
#2: Protein General transcription and DNA repair factor IIH subunit TFB4 / TFIIH subunit TFB4 / RNA polymerase II transcription factor B 34 kDa subunit / RNA polymerase II ...TFIIH subunit TFB4 / RNA polymerase II transcription factor B 34 kDa subunit / RNA polymerase II transcription factor B p34 subunit / RNA polymerase II transcription factor B subunit 4


Mass: 37506.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q12004
#5: Protein General transcription and DNA repair factor IIH subunit TFB2 / TFIIH subunit TFB2 / RNA polymerase II transcription factor B 52 kDa subunit / RNA polymerase II ...TFIIH subunit TFB2 / RNA polymerase II transcription factor B 52 kDa subunit / RNA polymerase II transcription factor B p52 subunit / RNA polymerase II transcription factor B subunit 2


Mass: 58602.312 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q02939
#7: Protein General transcription and DNA repair factor IIH subunit SSL1 / TFIIH subunit SSL1 / RNA polymerase II transcription factor B subunit SSL1 / TFB subunit SSL1 / ...TFIIH subunit SSL1 / RNA polymerase II transcription factor B subunit SSL1 / TFB subunit SSL1 / Suppressor of stem-loop protein 1


Mass: 52370.035 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q04673

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DNA repair helicase ... , 2 types, 2 molecules 70

#3: Protein DNA repair helicase RAD25 / General transcription and DNA repair factor IIH subunit RAD25 / TFIIH subunit RAD25 / Suppressor of ...General transcription and DNA repair factor IIH subunit RAD25 / TFIIH subunit RAD25 / Suppressor of stem-loop mutation 2


Mass: 95461.664 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q00578, DNA helicase
#6: Protein DNA repair helicase RAD3 / General transcription and DNA repair factor IIH subunit RAD3 / TFIIH subunit RAD3


Mass: 89899.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P06839, DNA helicase

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Protein , 1 types, 1 molecules 5

#4: Protein RNA polymerase II transcription factor B subunit 5 / General transcription and DNA repair factor IIH subunit TFB5 / TFIIH subunit TFB5


Mass: 8243.490 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q3E7C1

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Non-polymers , 2 types, 6 molecules

#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN

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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 TFIIH/Rad4-Rad23-Rad33 with AAF damaged DNA
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 420 kDa/nm / Experimental value: YES
Buffer solutionpH: 7.6
Details: Sample dialyzed for 30 minutes into above buffer prior to grid making
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMPotassium Acetate1
35 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was prepared by Grafix. Sample was monodispersed and well behaved by visual inspection
Specimen supportDetails: Pelco easyGLOW glow discharge apparatus / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE
Details: Grids were manually blotted for 2-3 seconds prior to plunge freezing

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Images collected with image shift
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6223
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.13CTF correction
7UCSF Chimeramodel fitting
8PHENIXmodel fitting
10PHENIXmodel refinement
11RELION3.0.8initial Euler assignment
12RELION3.0.8final Euler assignment
14RELION3D reconstruction
Image processingDetails: Camera operated in super resolution mode.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1159858
Details: Particles picked by elliptical blob picking in cryoSPARC
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56101 / Algorithm: FOURIER SPACE
Details: Final reconstruction performed in RELION. Map locally filtered to FSC=0.5 by blocres
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
15OQJ

5oqj

1
26NMI

6nmi

1

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