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- PDB-6nmi: Cryo-EM structure of the human TFIIH core complex -

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Entry
Database: PDB / ID: 6nmi
TitleCryo-EM structure of the human TFIIH core complex
Components
  • (General transcription and DNA repair factor IIH helicase subunit ...) x 2
  • (General transcription factor IIH subunit ...) x 5
  • CDK-activating kinase assembly factor MAT1
KeywordsTRANSCRIPTION / transcription / DNA repair / helicase / multiprotein complex
Function / homologyTFIIH subunit TTDA/Tfb5 / RNA Polymerase II HIV Promoter Escape / HIV Transcription Initiation / TFIIH subunit Tfb4/GTF2H3 / Transcription factor TFIIH subunit p52/Tfb2 / Cdk-activating kinase assembly factor MAT1/Tfb3 / Ubiquitin interacting motif / Zinc finger, RING-type / Transcription of the HIV genome / Cdk-activating kinase assembly factor MAT1, centre ...TFIIH subunit TTDA/Tfb5 / RNA Polymerase II HIV Promoter Escape / HIV Transcription Initiation / TFIIH subunit Tfb4/GTF2H3 / Transcription factor TFIIH subunit p52/Tfb2 / Cdk-activating kinase assembly factor MAT1/Tfb3 / Ubiquitin interacting motif / Zinc finger, RING-type / Transcription of the HIV genome / Cdk-activating kinase assembly factor MAT1, centre / Formation of HIV-1 elongation complex containing HIV-1 Tat / Tat-mediated elongation of the HIV-1 transcript / NoRC negatively regulates rRNA expression / Formation of Incision Complex in GG-NER / Dual Incision in GG-NER / RNA Polymerase II Pre-transcription Events / Zinc finger, RING/FYVE/PHD-type / Zinc finger, RING-type, conserved site / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Transcription factor Tfb2 (p52) C-terminal domain / Formation of HIV elongation complex in the absence of HIV Tat / Formation of the Early Elongation Complex / Formation of RNA Pol II elongation complex / Ubiquitin-interacting motif (UIM) domain profile. / Zinc finger RING-type profile. / Zinc finger RING-type signature. / Zinc finger, C3HC4 type (RING finger) / TFB5-like superfamily / CDK-activating kinase assembly factor MAT1 / Transcription factor TFIIH complex subunit Tfb5 / Transcription factor Tfb4 / Transcription factor Tfb2 / Transcription factor Tfb2, C-terminal domain / von Willebrand factor A-like domain superfamily / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / Formation of TC-NER Pre-Incision Complex / Formation of the HIV-1 Early Elongation Complex / Cyclin D associated events in G1 / RNA Polymerase II Promoter Escape / RNA Polymerase I Chain Elongation / mRNA Capping / Cyclin A:Cdk2-associated events at S phase entry / Cyclin A/B1/B2 associated events during G2/M transition / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase I Transcription Termination / RNA Polymerase II Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Transcription Elongation / Cyclin E associated events during G1/S transition / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Pol II CTD phosphorylation and interaction with CE / TP53 Regulates Transcription of DNA Repair Genes / Gap-filling DNA repair synthesis and ligation in TC-NER / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Dual incision in TC-NER / RNA Polymerase I Transcription Initiation / cyclin-dependent protein kinase activating kinase holoenzyme complex / ventricular system development / negative regulation of DNA helicase activity / core TFIIH complex portion of holo TFIIH complex / adult heart development / transcription factor TFIIH core complex / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity / phosphorylation of RNA polymerase II C-terminal domain / transcription elongation from RNA polymerase I promoter / transcription initiation from RNA polymerase I promoter / rDNA binding / termination of RNA polymerase I transcription / RNA polymerase II general transcription initiation factor activity / ATPase activator activity / nucleotide-excision repair, preincision complex stabilization / transcription factor TFIID complex / 7-methylguanosine mRNA capping / transcriptional preinitiation complex / regulation of cyclin-dependent protein serine/threonine kinase activity / transcription elongation from RNA polymerase II promoter / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / nucleotide-excision repair, DNA incision, 5'-to lesion / nucleotide-excision repair / nucleotide-excision repair, DNA incision / rRNA processing / cellular response to gamma radiation / G1/S transition of mitotic cell cycle / transcription-coupled nucleotide-excision repair / response to calcium ion / positive regulation of smooth muscle cell proliferation / protein-containing complex assembly / transcription by RNA polymerase II / double-stranded DNA binding / protein N-terminus binding / transcription initiation from RNA polymerase II promoter / nuclear speck / cell population proliferation / regulation of transcription by RNA polymerase II / DNA repair / nucleolus / protein phosphorylation / negative regulation of apoptotic process
Function and homology information
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.7 Å resolution
AuthorsGreber, B.J. / Toso, D. / Fang, J. / Nogales, E.
CitationJournal: Elife / Year: 2019
Title: The complete structure of the human TFIIH core complex.
Authors: Basil J Greber / Daniel B Toso / Jie Fang / Eva Nogales
Abstract: Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is ...Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 Å resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 10, 2019 / Release: Mar 13, 2019

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Assembly

Deposited unit
A: General transcription and DNA repair factor IIH helicase subunit XPB
B: General transcription and DNA repair factor IIH helicase subunit XPD
C: General transcription factor IIH subunit 1, p62
D: General transcription factor IIH subunit 4, p52
E: General transcription factor IIH subunit 2, p44
F: General transcription factor IIH subunit 3, p34
G: General transcription factor IIH subunit 5, p8
H: CDK-activating kinase assembly factor MAT1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)389,38915
Polyers388,6458
Non-polymers7447
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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General transcription and DNA repair factor IIH helicase subunit ... , 2 types, 2 molecules AB

#1: Protein/peptide General transcription and DNA repair factor IIH helicase subunit XPB


Mass: 74233.492 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: DNA helicase
#2: Protein/peptide General transcription and DNA repair factor IIH helicase subunit XPD


Mass: 86417.766 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa

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General transcription factor IIH subunit ... , 5 types, 5 molecules CDEFG

#3: Protein/peptide General transcription factor IIH subunit 1, p62


Mass: 57789.000 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa
#4: Protein/peptide General transcription factor IIH subunit 4, p52 / Basic transcription factor 2 52 kDa subunit / BTF2 p52 / General transcription factor IIH polypeptide 4 / TFIIH basal transcription factor complex p52 subunit


Mass: 52245.156 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: Q92759
#5: Protein/peptide General transcription factor IIH subunit 2, p44


Mass: 39609.098 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa
#6: Protein/peptide General transcription factor IIH subunit 3, p34 / p34 / BASIC TRANSCRIPTION FACTOR 2 34 KDA SUBUNIT / BTF2 P34 / GENERAL TRANSCRIPTION FACTOR IIH POLYPEPTIDE 3 / TFIIH BASAL TRANSCRIPTION FACTOR COMPLEX P34 SUBUNIT


Mass: 34416.008 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: Q13889
#7: Protein/peptide General transcription factor IIH subunit 5, p8 / General transcription factor IIH polypeptide 5 / TFB5 ortholog / TFIIH basal transcription factor complex TTD-A subunit


Mass: 8060.362 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: Q6ZYL4

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Protein/peptide , 1 types, 1 molecules H

#8: Protein/peptide CDK-activating kinase assembly factor MAT1 / CDK7/cyclin-H assembly factor / Cyclin-G1-interacting protein / Menage a trois / RING finger protein 66 / RING finger protein MAT1 / p35 / p36


Mass: 35873.965 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: P51948

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Non-polymers , 2 types, 7 molecules

#9: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Formula: Fe4S4 / Iron–sulfur cluster
#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Formula: Zn / Zinc

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Details

Sequence detailsThe full sequence for General transcription factor IIH subunit 1, p62 ...The full sequence for General transcription factor IIH subunit 1, p62 MATSSEEVLLIVKKVRQKKQDGALYLMAERIAWAPEGKDRFTISHMYADIKCQKISPEGK AKIQLQLVLHAGDTTNFHFSNESTAVKERDAVKDLLQQLLPKFKRKANKELEEKNRMLQE DPVLFQLYKDLVVSQVISAEEFWANRLNVNATDSSSTSNHKQDVGISAAFLADVRPQTDG CNGLRYNLTSDIIESIFRTYPAVKMKYAENVPHNMTEKEFWTRFFQSHYFHRDRLNTGSK DLFAECAKIDEKGLKTMVSLGVKNPLLDLTALEDKPLDEGYGISSVPSASNSKSIKENSN AAIIKRFNHHSAMVLAAGLRKQEAQNEQTSEPSNMDGNSGDADCFQPAVKRAKLQESIEY EDLGKNNSVKTIALNLKKSDRYYHGPTPIQSLQYATSQDIINSFQSIRQEMEAYTPKLTQ VLSSSAASSTITALSPGGALMQGGTQQAINQMVPNDIQSELKHLYVAVGELLRHFWSCFP VNTPFLEEKVVKMKSNLERFQVTKLCPFQEKIRRQYLSTNLVSHIEEMLQTAYNKLHTWQ SRRLMKKT General transcription and DNA repair factor IIH helicase subunit XPB MGKRDRADRDKKKSRKRHYEDEEDDEEDAPGNDPQEAVPSAAGKQVDESGTKVDEYGAKD YRLQMPLKDDHTSRPLWVAPDGHIFLEAFSPVYKYAQDFLVAIAEPVCRPTHVHEYKLTA YSLYAAVSVGLQTSDITEYLRKLSKTGVPDGIMQFIKLCTVSYGKVKLVLKHNRYFVESC HPDVIQHLLQDPVIRECRLRNSEGEATELITETFTSKSAISKTAESSGGPSTSRVTDPQG KSDIPMDLFDFYEQMDKDEEEEEETQTVSFEVKQEMIEELQKRCIHLEYPLLAEYDFRND SVNPDINIDLKPTAVLRPYQEKSLRKMFGNGRARSGVIVLPCGAGKSLVGVTAACTVRKR CLVLGNSAVSVEQWKAQFKMWSTIDDSQICRFTSDAKDKPIGCSVAISTYSMLGHTTKRS WEAERVMEWLKTQEWGLMILDEVHTIPAKMFRRVLTIVQAHCKLGLTATLVREDDKIVDL NFLIGPKLYEANWMELQNNGYIAKVQCAEVWCPMSPEFYREYVAIKTKKRILLYTMNPNK FRACQFLIKFHERRNDKIIVFADNVFALKEYAIRLNKPYIYGPTSQGERMQILQNFKHNP KINTIFISKVGDTSFDLPEANVLIQISSHGGSRRQEAQRLGRVLRAKKGMVAEEYNAFFY SLVSQDTQEMAYSTKRQRFLVDQGYSFKVITKLAGMEEEDLAFSTKEEQQQLLQKVLAAT DLDAEEEVVAGEFGSRSSQASRRFGTMSSMSGADDTVYMEYHSSRSKAPSKHVHPLFKRF RK General transcription factor IIH subunit 1 MATSSEEVLLIVKKVRQKKQDGALYLMAERIAWAPEGKDRFTISHMYADIKCQKISPEGK AKIQLQLVLHAGDTTNFHFSNESTAVKERDAVKDLLQQLLPKFKRKANKELEEKNRMLQE DPVLFQLYKDLVVSQVISAEEFWANRLNVNATDSSSTSNHKQDVGISAAFLADVRPQTDG CNGLRYNLTSDIIESIFRTYPAVKMKYAENVPHNMTEKEFWTRFFQSHYFHRDRLNTGSK DLFAECAKIDEKGLKTMVSLGVKNPLLDLTALEDKPLDEGYGISSVPSASNSKSIKENSN AAIIKRFNHHSAMVLAAGLRKQEAQNEQTSEPSNMDGNSGDADCFQPAVKRAKLQESIEY EDLGKNNSVKTIALNLKKSDRYYHGPTPIQSLQYATSQDIINSFQSIRQEMEAYTPKLTQ VLSSSAASSTITALSPGGALMQGGTQQAINQMVPNDIQSELKHLYVAVGELLRHFWSCFP VNTPFLEEKVVKMKSNLERFQVTKLCPFQEKIRRQYLSTNLVSHIEEMLQTAYNKLHTWQ SRRLMKKT General transcription and DNA repair factor IIH helicase subunit XPD MKLNVDGLLVYFPYDYIYPEQFSYMRELKRTLDAKGHGVLEMPSGTGKTVSLLALIMAYQ RAYPLEVTKLIYCSRTVPEIEKVIEELRKLLNFYEKQEGEKLPFLGLALSSRKNLCIHPE VTPLRFGKDVDGKCHSLTASYVRAQYQHDTSLPHCRFYEEFDAHGREVPLPAGIYNLDDL KALGRRQGWCPYFLARYSILHANVVVYSYHYLLDPKIADLVSKELARKAVVVFDEAHNID NVCIDSMSVNLTRRTLDRCQGNLETLQKTVLRIKETDEQRLRDEYRRLVEGLREASAARE TDAHLANPVLPDEVLQEAVPGSIRTAEHFLGFLRRLLEYVKWRLRVQHVVQESPPAFLSG LAQRVCIQRKPLRFCAERLRSLLHTLEITDLADFSPLTLLANFATLVSTYAKGFTIIIEP FDDRTPTIANPILHFSCMDASLAIKPVFERFQSVIITSGTLSPLDIYPKILDFHPVTMAT FTMTLARVCLCPMIIGRGNDQVAISSKFETREDIAVIRNYGNLLLEMSAVVPDGIVAFFT SYQYMESTVASWYEQGILENIQRNKLLFIETQDGAETSVALEKYQEACENGRGAILLSVA RGKVSEGIDFVHHYGRAVIMFGVPYVYTQSRILKARLEYLRDQFQIRENDFLTFDAMRHA AQCVGRAIRGKTDYGLMVFADKRFARGDKRGKLPRWIQEHLTDANLNLTVDEGVQVAKYF LRQMAQPFHREDQLGLSLLSLEQLESEETLKRIEQIAQQL

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Transcription factor IIH (TFIIH) / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: NATURAL
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameBuffer ID
120 mMHepes-KOH1
2150 mMSodium chloride1
35 mMMagnesium chloride1
40.015 %NP40 substitute1
51.5 %Glycerol1
62 %Trehalose1
SpecimenConc.: 0.0049 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Gatan Solarus / Grid material: COPPER / Grid mesh size: 400 / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins
Details: A thin film of continuous carbon was floated onto Protochips C-flat CF-4/2 holey carbon grids and glow discharged or plasma cleaned before application of 4 uL of sample solution.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS / Details: Residual beam tilt corrected in RELION 3.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 43478 / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.25 sec. / Electron dose: 50 e/Å2
Details: Images were collected as dose-fractionated movie frames (33 or 50 frames per exposure).
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 7 / Number of real images: 21437
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Phase plate: A Thermo Fisher Scientific Volta Phase Plate was used.

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Processing

SoftwareName: PHENIX / Version: 1.14_3211: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4GctfCTF correctionCTF determination
5RELION3CTF correctionCTF correction
8UCSF Chimeramodel fittingInitial fitting
9Cootmodel fittingRebuilding
10Omodel fittingRebuilding
12RELION3initial Euler assignment
13RELION3final Euler assignment
14RELION3classification
15RELION33D reconstruction
16PHENIXmodel refinementReal Space Refine
CTF correctionDetails: CTF correction during 3D reconstruction in RELION 3.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 138659 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingDetails: Reference structures and homology models were docked and subsequently completely rebuilt according to the density. Parts of the structure were traced de novo.
Ref protocol: RIGID BODY FIT / Ref space: REAL
Atomic model building
IDPDB-ID 3D fitting ID
15OF41
24ERN1
35OQJ1
41G251
55O851
62DII1
72JNJ1

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