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- PDB-5of4: The cryo-EM structure of human TFIIH -

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Entry
Database: PDB / ID: 5of4
TitleThe cryo-EM structure of human TFIIH
DescriptorMAT1
Unassigned secondary structure elements.
(TFIIH basal transcription factor complex helicase ...) x 2
(General transcription factor IIH subunit ...) x 4
(Unassigned secondary structure elements ...) x 2
KeywordsTRANSCRIPTION / transcription initiation / DNA repair / multiprotein complex / kinase / helicase
Specimen sourceHomo sapiens / human
MethodElectron microscopy (4.4 Å resolution / Particle / Single particle)
AuthorsGreber, B.J. / Nguyen, T.H.D. / Fang, J. / Afonine, P.V. / Adams, P.D. / Nogales, E.
CitationNature, 2017, 549, 414-417

Nature, 2017, 549, 414-417 Yorodumi Papers
The cryo-electron microscopy structure of human transcription factor IIH.
Basil J Greber / Thi Hoang Duong Nguyen / Jie Fang / Pavel V Afonine / Paul D Adams / Eva Nogales

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 10, 2017 / Release: Sep 13, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Sep 13, 2017Structure modelrepositoryInitial release
1.1Sep 27, 2017Structure modelDatabase referencescitation_citation.pdbx_database_id_PubMed / _citation.title
1.2Oct 4, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: TFIIH basal transcription factor complex helicase XPB subunit,XPB,TFIIH basal transcription factor complex helicase XPB subunit
B: TFIIH basal transcription factor complex helicase XPD subunit
D: General transcription factor IIH subunit 4,p52,General transcription factor IIH subunit 4
E: General transcription factor IIH subunit 2
F: General transcription factor IIH subunit 3
G: General transcription factor IIH subunit 5
H: MAT1
Z: Unassigned secondary structure elements.
Y: Unassigned secondary structure elements (p52 region)
X: Unassigned secondary structure elements (XPB NTE region)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)306,61811
Polyers306,26710
Non-polymers3521
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)18220
ΔGint (kcal/M)-129
Surface area (Å2)127880
MethodPISA

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Components

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TFIIH basal transcription factor complex helicase ... , 2 types, 2 molecules AB

#1: Polypeptide(L)TFIIH basal transcription factor complex helicase XPB subunit,XPB,TFIIH basal transcription factor complex helicase XPB subunit / Basic transcription factor 2 89 kDa subunit / BTF2 p89 / DNA excision repair protein ERCC-3 / DNA repair protein complementing XP-B cells / TFIIH basal transcription factor complex 89 kDa subunit / TFIIH p89 / Xeroderma pigmentosum group B-complementing protein


Mass: 62426.047 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens / References: UniProt: P19447, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

#2: Polypeptide(L)TFIIH basal transcription factor complex helicase XPD subunit / Basic transcription factor 2 80 kDa subunit / BTF2 p80 / CXPD / DNA excision repair protein ERCC-2 / DNA repair protein complementing XP-D cells / TFIIH basal transcription factor complex 80 kDa subunit / TFIIH p80 / Xeroderma pigmentosum group D-complementing protein


Mass: 87021.078 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens / References: UniProt: P18074, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

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General transcription factor IIH subunit ... , 4 types, 4 molecules DEFG

#3: Polypeptide(L)General transcription factor IIH subunit 4,p52,General transcription factor IIH subunit 4 / Basic transcription factor 2 52 kDa subunit / BTF2 p52 / General transcription factor IIH polypeptide 4 / TFIIH basal transcription factor complex p52 subunit


Mass: 9875.401 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens / References: UniProt: Q92759

Cellular component

Molecular function

Biological process

  • 7-methylguanosine mRNA capping (GO: 0006370)
  • DNA repair (GO: 0006281)
  • global genome nucleotide-excision repair (GO: 0070911)
  • nucleotide-excision repair (GO: 0006289)
  • nucleotide-excision repair, DNA duplex unwinding (GO: 0000717)
  • nucleotide-excision repair, DNA incision (GO: 0033683)
  • nucleotide-excision repair, DNA incision, 3'-to lesion (GO: 0006295)
  • nucleotide-excision repair, DNA incision, 5'-to lesion (GO: 0006296)
  • nucleotide-excision repair, preincision complex assembly (GO: 0006294)
  • nucleotide-excision repair, preincision complex stabilization (GO: 0006293)
  • phosphorylation of RNA polymerase II C-terminal domain (GO: 0070816)
  • termination of RNA polymerase I transcription (GO: 0006363)
  • transcription elongation from RNA polymerase I promoter (GO: 0006362)
  • transcription elongation from RNA polymerase II promoter (GO: 0006368)
  • transcription from RNA polymerase II promoter (GO: 0006366)
  • transcription initiation from RNA polymerase I promoter (GO: 0006361)
  • transcription initiation from RNA polymerase II promoter (GO: 0006367)
  • transcription-coupled nucleotide-excision repair (GO: 0006283)
#4: Polypeptide(L)General transcription factor IIH subunit 2 / Basic transcription factor 2 44 kDa subunit / BTF2 p44 / General transcription factor IIH polypeptide 2 / TFIIH basal transcription factor complex p44 subunit


Mass: 44481.996 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens / References: UniProt: Q13888

Cellular component

Molecular function

Biological process

  • 7-methylguanosine mRNA capping (GO: 0006370)
  • G-protein coupled receptor internalization (GO: 0002031)
  • global genome nucleotide-excision repair (GO: 0070911)
  • nucleotide-excision repair (GO: 0006289)
  • nucleotide-excision repair, DNA duplex unwinding (GO: 0000717)
  • nucleotide-excision repair, DNA incision (GO: 0033683)
  • nucleotide-excision repair, DNA incision, 3'-to lesion (GO: 0006295)
  • nucleotide-excision repair, DNA incision, 5'-to lesion (GO: 0006296)
  • nucleotide-excision repair, preincision complex assembly (GO: 0006294)
  • nucleotide-excision repair, preincision complex stabilization (GO: 0006293)
  • protein phosphorylation (GO: 0006468)
  • regulation of transcription from RNA polymerase II promoter (GO: 0006357)
  • response to UV (GO: 0009411)
  • termination of RNA polymerase I transcription (GO: 0006363)
  • transcription elongation from RNA polymerase I promoter (GO: 0006362)
  • transcription elongation from RNA polymerase II promoter (GO: 0006368)
  • transcription from RNA polymerase II promoter (GO: 0006366)
  • transcription initiation from RNA polymerase I promoter (GO: 0006361)
  • transcription initiation from RNA polymerase II promoter (GO: 0006367)
  • transcription-coupled nucleotide-excision repair (GO: 0006283)
#5: Polypeptide(L)General transcription factor IIH subunit 3 / 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 / References: UniProt: Q13889

Cellular component

Molecular function

Biological process

  • 7-methylguanosine mRNA capping (GO: 0006370)
  • DNA repair (GO: 0006281)
  • global genome nucleotide-excision repair (GO: 0070911)
  • nucleotide-excision repair (GO: 0006289)
  • nucleotide-excision repair, DNA duplex unwinding (GO: 0000717)
  • nucleotide-excision repair, DNA incision (GO: 0033683)
  • nucleotide-excision repair, DNA incision, 3'-to lesion (GO: 0006295)
  • nucleotide-excision repair, DNA incision, 5'-to lesion (GO: 0006296)
  • nucleotide-excision repair, preincision complex assembly (GO: 0006294)
  • nucleotide-excision repair, preincision complex stabilization (GO: 0006293)
  • phosphorylation of RNA polymerase II C-terminal domain (GO: 0070816)
  • termination of RNA polymerase I transcription (GO: 0006363)
  • transcription elongation from RNA polymerase I promoter (GO: 0006362)
  • transcription elongation from RNA polymerase II promoter (GO: 0006368)
  • transcription from RNA polymerase II promoter (GO: 0006366)
  • transcription initiation from RNA polymerase I promoter (GO: 0006361)
  • transcription initiation from RNA polymerase II promoter (GO: 0006367)
  • transcription-coupled nucleotide-excision repair (GO: 0006283)
#6: Polypeptide(L)General transcription factor IIH subunit 5 / 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 / References: UniProt: Q6ZYL4

Cellular component

Molecular function

Biological process

  • 7-methylguanosine mRNA capping (GO: 0006370)
  • cellular response to gamma radiation (GO: 0071480)
  • global genome nucleotide-excision repair (GO: 0070911)
  • nucleotide-excision repair (GO: 0006289)
  • nucleotide-excision repair, DNA duplex unwinding (GO: 0000717)
  • nucleotide-excision repair, DNA incision (GO: 0033683)
  • nucleotide-excision repair, DNA incision, 3'-to lesion (GO: 0006295)
  • nucleotide-excision repair, DNA incision, 5'-to lesion (GO: 0006296)
  • nucleotide-excision repair, preincision complex assembly (GO: 0006294)
  • nucleotide-excision repair, preincision complex stabilization (GO: 0006293)
  • regulation of transcription, DNA-templated (GO: 0006355)
  • rRNA processing (GO: 0006364)
  • termination of RNA polymerase I transcription (GO: 0006363)
  • transcription elongation from RNA polymerase I promoter (GO: 0006362)
  • transcription elongation from RNA polymerase II promoter (GO: 0006368)
  • transcription from RNA polymerase II promoter (GO: 0006366)
  • transcription initiation from RNA polymerase I promoter (GO: 0006361)
  • transcription initiation from RNA polymerase II promoter (GO: 0006367)
  • transcription-coupled nucleotide-excision repair (GO: 0006283)

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Polypeptide(L) , 2 types, 2 molecules HZ

#7: Polypeptide(L)MAT1


Mass: 10571.022 Da / Num. of mol.: 1 / Details: Sequence register unassigned. / Source: (natural) Homo sapiens
#8: Polypeptide(L)Unassigned secondary structure elements.


Mass: 22996.232 Da / Num. of mol.: 1 / Details: Sequence unassigned. / Source: (natural) Homo sapiens

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Unassigned secondary structure elements ... , 2 types, 2 molecules YX

#9: Polypeptide(L)Unassigned secondary structure elements (p52 region)


Mass: 19762.281 Da / Num. of mol.: 1 / Details: Sequence unassigned / Source: (natural) Homo sapiens
#10: Polypeptide(L)Unassigned secondary structure elements (XPB NTE region)


Mass: 6656.196 Da / Num. of mol.: 1 / Source: (natural) Homo sapiens

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Non-polymers , 1 types, 1 molecules

#11: ChemicalChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Formula: Fe4S4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1TFIIHCOMPLEX1, 2, 3, 4, 5, 6, 7, 8, 9, 100NATURAL
2TFIIH core complexCOMPLEX1, 2, 3, 4, 5, 6, 8, 9, 101NATURAL
3TFIIH CDK-activating kinase (CAK) subcomplexCOMPLEX71NATURAL
Molecular weight
IDValueUnitsEntity assembly IDExperimental value
10.49MEGADALTONS1NO
21NO
31NO
13
Source (natural)
IDEntity assembly IDNcbi tax IDOrganismCellular location
219606Homo sapiens
329606Homo sapiensNucleus
439606Homo sapiensNucleus
Buffer solutionpH: 7.8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mMHEPES-KOH1
2150mMpotassium chlorideKCl1
35mMmagnesium chlorideMgCl21
40.5mMDTT1
52%trehalose1
61.5%glycerol1
70.015%NP-40 substitute1
SpecimenConc.: 0.0049 mg/ml
Details: Natively purified complex at approx. 10 nM concentration
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-flat-4/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278.15 kelvins / Details: 3-4 minute incubation, 2 second blot

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 37879 / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 8.7 sec. / Electron dose: 40 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.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 4 / Number of real images: 8300
Image scansSampling size: 5 microns / Dimension width: 3840 / Dimension height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategoryImaging IDDetailsImage processing IDFitting ID
2LeginonIMAGE ACQUISITION1
4CTFFIND4CTF CORRECTIONrun from within RELION1
7UCSF ChimeraMODEL FITTING1
8OMODEL FITTING1
9CootMODEL FITTING1
11RELION1.4INITIAL EULER ASSIGNMENT1
12RELION1.4FINAL EULER ASSIGNMENT1
13RELION1.4CLASSIFICATION1
14RELION1.4RECONSTRUCTION1
15PHENIXdevelopment versionsMODEL REFINEMENTreal space refinement1
16REFMAC5.9MODEL REFINEMENTreciprocal space refinement1
Image processingDetails: Micrographs were inspected for quality of Thon rings and ice contamination. Poor micrographs were rejected.
CTF correctionDetails: Correction in RELION based on values determined in CTFFIND4.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Initial rounds of particle selection using DogPicker within APPION to generate templates for subsequent RELION autopicking.
Number of particles selected: 1500000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 122900 / Algorithm: FOURIER SPACE / Details: RELION 3D auto-refinement (gold standard). / Number of class averages: 3 / Symmetry type: POINT
Atomic model buildingDetails: Initial model assembled from high-resolution structures and homology models. Subsequently rebuilt in O and COOT, refined into the cryo-EM map using PHENIX and REFMAC, and fully validated.
Ref protocol: RIGID BODY FIT / Ref space: REAL
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcDetailsR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLOverall ESU RSolvent ion probe radiiSolvent shrinkage radiiSolvent vdw probe radiiStereochemistry target valuesSolvent model details
1193.578-4.69-0.800.57-3.42-2.548.110.939HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.335280.335284.40166.3282085100.0054.8490.6681.6790.800.801.20MAXIMUM LIKELIHOOD WITH PHASESMASK
ELECTRON MICROSCOPY
Number of atoms included #1Total: 17200
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0210.01917421
ELECTRON MICROSCOPYr_bond_other_d0.0030.02016607
ELECTRON MICROSCOPYr_angle_refined_deg2.1401.94023691
ELECTRON MICROSCOPYr_angle_other_deg1.2293.00037892
ELECTRON MICROSCOPYr_dihedral_angle_1_deg9.7835.0002411
ELECTRON MICROSCOPYr_dihedral_angle_2_deg26.45223.678609
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.55515.0002393
ELECTRON MICROSCOPYr_dihedral_angle_4_deg14.40615.00099
ELECTRON MICROSCOPYr_chiral_restr0.1120.2002879
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.02020092
ELECTRON MICROSCOPYr_gen_planes_other0.0030.0203515
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it36.53018.9359800
ELECTRON MICROSCOPYr_mcbond_other36.52918.9369799
ELECTRON MICROSCOPYr_mcangle_it58.66528.18312159
ELECTRON MICROSCOPYr_mcangle_other58.66328.18112160
ELECTRON MICROSCOPYr_scbond_it39.26720.9657621
ELECTRON MICROSCOPYr_scbond_other39.26520.9637622
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other64.30430.37511521
ELECTRON MICROSCOPYr_long_range_B_refined89.08269008
ELECTRON MICROSCOPYr_long_range_B_other89.08269009
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 4.4 Å / R factor R work: 0.459 / Lowest resolution: 4.514 Å / Number reflection R free: 0 / Number reflection R work: 6100 / Total number of bins used: 20 / Percent reflection obs: 1

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