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The cryo-electron microscopy structure of human transcription factor IIH

Nature volume 549, pages 414–417 (2017)Cite this article

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Abstract

Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription1. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair1. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair2, 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 initiation1,2. 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 physiology3. 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.

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Figure 1: Cryo-EM reconstruction of human TFIIH.
Figure 2: Structures of human XPB and XPD.
Figure 3: MAT1 forms a physical connection between all ATP consuming TFIIH moieties.
Figure 4: Conformational rearrangements of TFIIH.

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Acknowledgements

We thank S. Zheng and D. King for providing peptides and XPB monoclonal antibody, P. Grob for cryo-EM support, and T. Houweling and A. Chintangal for computing support. We thank A. B. Patel and R. K. Louder for discussions and help with data collection, and J. H. D. Cate for providing PyMOL scripts. We acknowledge the use of the LAWRENCIUM computing cluster at Lawrence Berkeley National Laboratory and the resources of the National Energy Research Scientific Computing Center, a Department of Energy Office of Science user facility supported by the Office of Science of the US Department of Energy under contract number DE-AC02-05CH11231. This work was funded through NIGMS grants R01-GM63072 to E.N. and P01-GM063210 to P.D.A. B.J.G. was supported by fellowships from the Swiss National Science Foundation (projects P300PA_160983, P300PA_174355), and T.H.D.N. is a University of California, Berkeley Miller Fellow. E.N. is a Howard Hughes medical investigator.

Author information

Authors and Affiliations

  1. California Institute for Quantitative Biology (QB3), University of California, Berkeley, 94720, California, USA

    Basil J. Greber, Thi Hoang Duong Nguyen & Eva Nogales

  2. Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Basil J. Greber, Thi Hoang Duong Nguyen, Pavel V. Afonine, Paul D. Adams & Eva Nogales

  3. Miller Institute for Basic Research in Science, University of California, Berkeley, 94720, California, USA

    Thi Hoang Duong Nguyen

  4. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Jie Fang & Eva Nogales

  5. Department of Bioengineering, University of California, Berkeley, 94720, California, USA

    Paul D. Adams

  6. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Eva Nogales

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  1. Basil J. Greber
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Contributions

E.N. directed the study. J.F. performed HeLa cell culture and prepared TFIIH. B.J.G. performed cryo-EM specimen preparation, data collection, data processing, and initial model building. B.J.G. and T.H.D.N. built the final model. B.J.G. and T.H.D.N. performed coordinate refinement, supported by P.V.A. in the laboratory of P.D.A. B.J.G. wrote the initial draft of the manuscript and all authors contributed to the final version.

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Correspondence to Eva Nogales.

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Reviewer Information Nature thanks S. Hahn, O. Llorca and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Data processing and cryo-EM map refinement.

a, Schematic of the data processing and classification procedure. See text and Methods for details. b, Representative class averages obtained during the reference-free 2D classification step in a (taken from dataset 4). c, Comparison of the best-resolved densities of intermediate stage classified maps (asterisk in a) from dataset 2 (left), dataset 1 (middle), and the final map (shown for comparison, right). The XPD coordinate model is shown in green. Owing to the lack of β-strand separation, dataset 1 was discarded from the final refinement (see Methods for details).

Extended Data Figure 2 Resolution estimation and focused classification.

a, FSC curves. Black: FSC curve for the cryo-EM map of the TFIIH core complex, computed from auto-masked half-maps in RELION38, indicating a resolution of 4.4 Å according to the gold-standard FSC = 0.143 criterion42,62. Red: FSC curve between the cryo-EM map and the refined atomic coordinates. The coordinate refinement used data until 4.4 Å; meaningful correlation between the model and the map extends to 4.3 Å (FSC = 0.5 criterion). b, Local resolution estimation using the BLOCRES command of the BSOFT package63. Most of the core of the density of the cryo-EM reconstruction is resolved at 4 Å or better. c, d, Signal-subtracted 3D classifications to resolve heterogeneity near the MAT1 binding site on XPD (c) and of the XPD 4FeS domain (d). The 122,900 particles used to reconstruct the TFIIH cryo-EM map at 4.4 Å resolution were used for this step. Three-dimensional classes obtained from signal-subtracted particles are shown on the left, overlaid with a transparent depiction of the entire TFIIH map for orientation. Boxed classes were refined (two classes were combined in d). The cryo-EM map obtained by refining the non-signal-subtracted particles corresponding to the particles assigned to the boxed 3D classes is shown on the right. e, f, FSC curves for the reconstructions obtained after signal-subtracted 3D classification (c, d) for the XPD 4FeS domain (e) and the additional density near the XPD ARCH domain (f).

Extended Data Figure 3 Coordinate refinement and atomic model of TFIIH.

a–f, Examples of the cryo-EM density of the TFIIH core complex, showing α-helices with side chain densities (a), separation of β-strands (b, c), and density for large side chains (d–f). g, Refinement table summarizing the key statistics from the atomic coordinate refinement. h, Representation of the refined atomic coordinate model coloured according to B-factors (top), and viewed in section (bottom), showing lower B-factors in the better-ordered core of the complex, as expected. i, Model validation using data-halfsets. The refined model was refined against one of the gold-standard half-maps (work map). The FSC curve (FSCwork) between the refined model and the work map is shown in green; the FSC curve (FSCtest) between the model and the other half-map (test map, not used in refinement) is shown in blue. The half-maps were summed and the model was also refined against the summed half-maps (black FSC curve, FSCfull).

Extended Data Figure 4 Domain architectures and secondary structure predictions of TFIIH components.

Domain organization and secondary structure prediction of the protein subunits of the TFIIH core complex and MAT1. Proteins and domains with known 3D structures (human proteins) are shown as coloured areas (colours used correspond to those used for molecular depictions) and labelled. All of these are modelled in our molecular structure, except those regions in p44 and p62 marked with an asterisk and the MAT1 ring finger domain. Dark green bars on the grey background indicate predicted α-helices, dark blue bars represent predicted β-strands (predictions were obtained using PHYRE2 (ref. 45)). Only secondary structure elements predicted with high confidence are displayed. The longest predicted α-helix of the entire TFIIH complex is found in MAT1 and measures more than 50 residues (indicated with a schematic helix).

Extended Data Figure 5 Modelling of secondary structure elements into regions of previously unassigned density.

a, b, Unassigned density around the p34–p44–XPD region is shown in teal, around the p52–XPB NTE region in red, and near the p52 helical domain in gold. Densities are low-pass filtered to 6 Å for clarity. Some of these densities could be interpreted by placement of secondary structure elements and extended linkers. c, Overview of the molecular model after placement of secondary structures into unassigned densities. Remaining unassigned densities are shown in the same colours as in a and b and probably harbour parts of p62 and the zinc-binding domains of p34 and p44. Newly modelled elements are shown in cartoon representation in the same colours as the density regions into which they were modelled. Densities are low-pass filtered to 6 Å for clarity. d, Several α-helices could be placed near the hinge region between p34 and p44, although some density remains unassigned (shown as teal surface). e, Density and model for the three-helix bundle near the hinge region (d). f, In addition to the density of the hinge region (d), several linkers and α-helices wrap around the p34 von Willebrand factor A-like domain core, and a small domain (asterisk) is bound to its side. g, Secondary structure elements (red) modelled into unassigned density (red density in b) in the vicinity of the XPB RecA-like domains and the p52 CTD include the XPB NTE and DRD and a β-sheet tentatively assigned the p52 (see Fig. 2e). h, Density and model for one of the β-sheets of the XPB NTE. i, j, Mapping of CX-MS crosslinks7 (summarized in Supplementary Data Table 1) onto the structure of TFIIH. Crosslinks are colour-coded (black: Cα–Cα distance <30 Å; yellow: 30–35 Å; red: >35 Å). Intermolecular crosslinks are shown in i, intramolecular crosslinks in j.

Extended Data Figure 6 Analysis of the human XPB and XPD structures.

a, A cryo-EM density peak (dark blue surface) for the 4Fe4S cluster can be seen within the 4FeS domain of XPD. b, Comparison of the structure of the 4FeS domains in human (brown) and archaeal XPD (grey; PDB accession number 3CRV)10. The 4Fe4S cluster and coordinating cysteine residues of the human protein are shown in stick representation. c, On the back side of XPD, the 4Fe4S cluster is in proximity of the pore region, which may position it to participate in charge-transfer reactions with DNA passing through the pore8,10. d, Model for the possible path of DNA on XPD obtained by superimposing the DNA-bound structures of archaeal XPD enzymes (PDB accession numbers 4A15, 5H8W)11,12 and the HCV NS3 helicase (PDB accession number 1A1V)64. For the NS3 helicase, the two RecA-like domains were superposed individually. The DNA fragments shown in red suggest a possible path of substrate DNA in XPD from the pore between the ARCH and 4FeS domains towards RecA2. The helicase motifs10,16 are coloured on the basis of known function (blue: DNA binding; dark red: nucleotide binding and hydrolysis; yellow: coordination between ATPase and helicase activity). e, Same as d, but viewed from the back side of the pore (as in c). f, The p44 subunit interacts with XPD RecA2 near the conserved helicase elements IV and V (helicase elements indicated; XPD coloured as in d). g, h, The p44–XPD (g), eIF4G–eIF4A (PDB accession number 2VSX (ref. 14), h), and Gle1–Dbp5 (PDB accession number 3RRN (ref. 15), h) interactions occur on the same regions of RecA2 of the respective helicases. eIF4G and Gle1 additionally interact with RecA1 of eIF4A and Dbp5, respectively. i, j, Comparison of human XPB with the archaeal ssoRad54 SWI2/SNF2 ATPase (grey; bound double-stranded DNA in red; PDB accession number 1Z63)21. RecA2 of the SWI2/SNF2 ATPase was superposed individually on XPB owing to conformational differences. XPB shows clear homology to SWI2/SNF2 ATPases, including a domain insertion in ssoRad54 (light brown) that occurs in the same place as the XPB thumb motif (brown) (i) and the presence of a Gln/Asn-Trp motif in helicase motif Ia16 (j). k, XPB RecA2 is coloured using a spectrum from blue (N terminus) to red (C terminus). Black arrows (outward movement and rotation) indicate the domain motions required to achieve the arrangement observed for the RecA-like domains in an archaeal SWI2/SNF2 ATPase bound to DNA (PDB accession number 1Z63)21. This motion would lead to extensive breakage of protein interfaces in TFIIH (regions losing contact are indicated by red arrows in the right panel).

Extended Data Figure 7 Modelling of the DRD-like domain in human XPB.

a–i, Views of a fragment of PDB accession number 2BOP found by an unbiased molecular replacement search of the density eventually assigned to the XPB DRD-like domain (a–c, see Methods), the archaeal XPB DRD (PDB accession number 2FWR20; d–f), and the HARP1 domain (PDB accession number 4O66 (ref. 49); g–i) fitted into the TFIIH cryo-EM map. The fits reveal an excellent agreement of the β-sheet and the connecting α-helices with the density. j–l, The molecular model of TFIIH is shown in the same orientations as the above panels to show the structure of the human XPB DRD-like domain in the cryo-EM map. m, Comparison of the overall orientation of the DRD in archaeal XPB (cyan) and the DRD-like domain in human XPB (blue) with respect to their RecA-like domains. n, It has not been conclusively determined whether the DRD-like domain in human XPB binds DNA. A hypothetical model obtained by superposition of the MutS DRD bound to DNA (PDB accession number 1EWQ)65 onto the XPB DRD-like domain suggests that DRD-bound DNA would approach XPD in TFIIH. MAT1 overlaps with the DNA bound in this orientation, indicating that if the XPB DRD-like domain binds DNA as suggested by this model, it may occur in a context where MAT1 is not present in the complex, such as during NER.

Extended Data Figure 8 Analysis of MAT1 and the remainder of the CAK subcomplex.

a, Two negative-stain class averages showing clear density for the CAK subcomplex (top row) and schematic representation of the position of the CAK, MAT1, XPB, and XPD in these class averages (bottom row). b, Schematic representation of the position and range of motion of the CAK subcomplex inferred from 2D negative-stain class averages like those shown in a. This high degree of structural flexibility of the CAK subcomplex precludes its visualization in the high-resolution cryo-EM map of TFIIH. c, Negative-stain 3D reconstructions of TFIIH complexes including density for the CAK and data processing strategy used to obtain these reconstructions. All processing was done in RELION38. The coordinate model of TFIIH is fitted into three of the obtained classes. The long helix and helical bundle (Fig. 3a, b) localize to the region where the CAK and core subcomplexes interact. d, Unassigned density near the three-helix bundle of MAT1 is coloured in blue. The map shown was obtained by signal-subtracted classification43 for this region of XPD/MAT1 (Extended Data Fig. 2c) and subsequent refinement. e, Size comparison between the N-terminal MAT1 ring finger domain66 (PDB accession number 1G25) and the unassigned density near MAT1 and the XPD ARCH domain. f, g, Comparison of the density in the Pol II-PIC5 attributed to the CAK (light yellow) with the density observed in the negative-stain reconstructions of free TFIIH (pink). The CAK density in free TFIIH is close to the corresponding density in the context of the Pol II-PIC.

Extended Data Figure 9 Conformational rearrangements of TFIIH.

a, Contact areas between XPB (blue) and XPD (green) or p44 (red) are shown in space-filling representation to highlight the extent of the interactions. The view is otherwise identical to the close-up in Fig. 4a. Unassigned secondary structure elements (chain Z in the atomic model) are not shown for clarity. b, Coordinate model of free TFIIH shown in sphere representation. Colours as in a. MAT1 and unassigned secondary structure elements in chain Z are not shown for clarity and ease of comparison. c, Same as b, but atomic coordinates re-fitted into the cryo-EM map of TFIIH in the context of the Pol II-PIC5 (EMD-8131). No contacts from XPB to XPD and p44 are observed in this complex. d, The comparison of the cryo-EM maps of free TFIIH (teal) and TFIIH in the context of the Pol II-PIC5 (EMD-8131) shows that the dimensions of the two reconstructions are almost identical along the long axis of TFIIH (horizontal), but clearly differ along the short axis (vertical) owing to TFIIH opening within the DNA-engaged PIC. e, Model of free TFIIH in its cryo-EM map. f, Model of TFIIH in the Pol II-PIC map5 (PDB accession number 5IVW, EMD-8131).

Extended Data Table 1 Summary of components included in the TFIIH molecular model
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Supplementary Information

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Supplementary Data

This file contains an analysis of CX-MS data for human TFIIH. (XLSX 54 kb)

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Greber, B., Nguyen, T., Fang, J. et al. The cryo-electron microscopy structure of human transcription factor IIH. Nature 549, 414–417 (2017). https://doi.org/10.1038/nature23903

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