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- EMDB-8817: TFIIH reconstruction from particles sorted for the XPD FeS-cluste... -

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Basic information

Entry
Database: EMDB / ID: EMD-8817
TitleTFIIH reconstruction from particles sorted for the XPD FeS-cluster domain using signal-subtracted classification.
Map dataTFIIH reconstruction from particles sorted for the FeS-cluster domain using signal-subtracted classification.
Sample
  • Complex: Transcription factor IIH (TFIIH)
Function / homology
Function and homology information


MMXD complex / core TFIIH complex portion of holo TFIIH complex / positive regulation of DNA helicase activity / Cytosolic iron-sulfur cluster assembly / nucleotide-excision repair, DNA duplex unwinding / central nervous system myelin formation / positive regulation of mitotic recombination / hair cell differentiation / hair follicle maturation / nucleotide-excision repair factor 3 complex ...MMXD complex / core TFIIH complex portion of holo TFIIH complex / positive regulation of DNA helicase activity / Cytosolic iron-sulfur cluster assembly / nucleotide-excision repair, DNA duplex unwinding / central nervous system myelin formation / positive regulation of mitotic recombination / hair cell differentiation / hair follicle maturation / nucleotide-excision repair factor 3 complex / nucleotide-excision repair, preincision complex assembly / UV protection / CAK-ERCC2 complex / embryonic cleavage / 5'-3' DNA helicase activity / G protein-coupled receptor internalization / transcription factor TFIIH holo complex / transcription factor TFIIH core complex / 3'-5' DNA helicase activity / transcription preinitiation complex / RNA Polymerase I Transcription Termination / regulation of mitotic cell cycle phase transition / hematopoietic stem cell proliferation / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / RNA polymerase II general transcription initiation factor activity / spinal cord development / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / transcription factor TFIID complex / mRNA Capping / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / 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 / bone mineralization / erythrocyte maturation / ATPase activator activity / RNA Polymerase I Transcription Initiation / DNA topological change / transcription elongation by RNA polymerase I / intrinsic apoptotic signaling pathway by p53 class mediator / transcription-coupled nucleotide-excision repair / Tat-mediated elongation of the HIV-1 transcript / hematopoietic stem cell differentiation / Formation of HIV-1 elongation complex containing HIV-1 Tat / embryonic organ development / Formation of HIV elongation complex in the absence of HIV Tat / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / response to UV / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / RNA Polymerase II Pre-transcription Events / DNA helicase activity / extracellular matrix organization / post-embryonic development / insulin-like growth factor receptor signaling pathway / transcription elongation by RNA polymerase II / chromosome segregation / determination of adult lifespan / promoter-specific chromatin binding / transcription initiation at RNA polymerase II promoter / nucleotide-excision repair / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase I Promoter Escape / multicellular organism growth / Dual Incision in GG-NER / NoRC negatively regulates rRNA expression / protein localization / spindle / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / cellular response to gamma radiation / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / protein-macromolecule adaptor activity / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / response to oxidative stress / DNA helicase / in utero embryonic development / damaged DNA binding / transcription by RNA polymerase II / response to hypoxia / nuclear speck / positive regulation of apoptotic process / DNA repair / apoptotic process / nucleolus / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding
Similarity search - Function
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 / RAD3/XPD family / Helicase XPB/Ssl2 ...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 / RAD3/XPD family / Helicase XPB/Ssl2 / Helical and beta-bridge domain / ERCC3/RAD25/XPB helicase, C-terminal domain / Helicase XPB/Ssl2, N-terminal domain / Helical and beta-bridge domain / Helicase conserved C-terminal domain / ERCC3/RAD25/XPB C-terminal helicase / Transcription factor TFIIH subunit p52/Tfb2 / ATP-dependent helicase Rad3/Chl1-like / 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 / Helicase superfamily 1/2, DinG/Rad3-like / Helicase-like, DEXD box c2 type / ATP-dependent helicase, C-terminal / DEAD2 / Helicase superfamily 1/2, ATP-binding domain, DinG/Rad3-type / DEAD_2 / Helicase C-terminal domain / Superfamilies 1 and 2 helicase ATP-binding type-2 domain profile. / DEXDc2 / HELICc2 / 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 / 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
General transcription and DNA repair factor IIH helicase subunit XPD / General transcription and DNA repair factor IIH helicase subunit XPB / General transcription factor IIH subunit 2 / General transcription factor IIH subunit 3 / General transcription factor IIH subunit 5 / General transcription factor IIH subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsGreber BJ / Nguyen THD / Fang J / Afonine PV / Adams PD / Nogales E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM63072 United States
CitationJournal: Nature / Year: 2017
Title: The cryo-electron microscopy structure of human transcription factor IIH.
Authors: Basil J Greber / Thi Hoang Duong Nguyen / Jie Fang / Pavel V Afonine / Paul D Adams / Eva Nogales /
Abstract: Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits ...Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIH subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Additionally, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.
History
DepositionJul 10, 2017-
Header (metadata) releaseJul 26, 2017-
Map releaseOct 4, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8817.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTFIIH reconstruction from particles sorted for the FeS-cluster domain using signal-subtracted classification.
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.06940234 - 0.1495091
Average (Standard dev.)-0.0000080399 (±0.00466946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 337.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z337.920337.920337.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0690.150-0.000

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Supplemental data

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Sample components

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Entire : Transcription factor IIH (TFIIH)

EntireName: Transcription factor IIH (TFIIH)
Components
  • Complex: Transcription factor IIH (TFIIH)

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Supramolecule #1: Transcription factor IIH (TFIIH)

SupramoleculeName: Transcription factor IIH (TFIIH) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 490 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.0049 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
20.0 mMHEPES-KOH
150.0 mMpotassium chlorideKCl
5.0 mMmagnesium chlorideMgCl2
0.5 mMDTT
2.0 %trehalose
1.5 %glycerol
0.015 %NP-40 substitute
GridModel: Protochips C-flat 4/2 / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV / Details: 3-4 minute incubation, 2 second blot.
DetailsNatively purified complex at approx. 10 nM concentration

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 37879 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-30 / Number grids imaged: 4 / Number real images: 8300 / Average exposure time: 8.7 sec. / Average electron dose: 40.0 e/Å2
Details: 8300 micrographs collected in four session with identical acquisition settings. Sessions lasted 4, 2, 4, and 4 days and yielded 1200, 1700, 2800, and 2600 micrographs.

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Image processing

Particle selectionNumber selected: 1500000
Details: Initial rounds of particle selection using DogPicker within APPION to generate templates for subsequent RELION autopicking.
CTF correctionSoftware - Name: CTFFIND (ver. 4) / Software - details: run from within RELION
Details: Correction within RELION based on values determined in CTFFIND4.
Startup modelType of model: EMDB MAP
EMDB ID:

Details: Box and pixel size were adjusted before initial refinement.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Details: Maximum-likelihood 3D classification within RELION.
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 1.4)
Details: Signal-subtracted classification in RELION 1.4, using 6 classes.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4) / Details: Maximum-likelihood 3D refinement within RELION.
Final reconstructionNumber classes used: 2 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4)
Details: RELION 3D auto-refinement (gold standard). The final 3D classification used signal-subtracted particle images. The final reconstruction used the corresponding un-subtracted particle images.
Number images used: 88700
DetailsMicrographs were inspected for quality of Thon rings and ice contamination. Poor micrographs were rejected.

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Atomic model buiding 1

DetailsFitting of a refined coordinate model obtained from a 4.4 Angstrom reconstruction of TFIIH from the same initial dataset (PDB ID 5OF4).
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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