National Institutes of Health/National Institute of General Medical Sciences
R01-GM63072
米国
Swiss National Science Foundation
P300PA_160983
スイス
National Institutes of Health/National Institute of General Medical Sciences
P01-GM063210
米国
Swiss National Science Foundation
P300PA_174355
スイス
引用
ジャーナル: Elife / 年: 2019 タイトル: The complete structure of the human TFIIH core complex. 著者: Basil J Greber / Daniel B Toso / Jie Fang / Eva Nogales / 要旨: 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.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: 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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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
特殊光学系
位相板: VOLTA PHASE PLATE / エネルギーフィルター - 名称: GIF Quantum LS / エネルギーフィルター - スリット幅: 20 eV
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / 撮影したグリッド数: 7 / 実像数: 21437 / 平均露光時間: 8.25 sec. / 平均電子線量: 50.0 e/Å2 詳細: Images were collected as dose-fractionated movie frames (33 or 50 frames per exposure).
使用したクラス数: 1 / 想定した対称性 - 点群: C1 (非対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 4.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3) 詳細: The map resolution by FSC=0.143 is 4.1 Angstroms. The map was low-pass filtered to 4.5 Angstroms resolution to improve the interpretability of the region of interest. 使用した粒子像数: 81131
初期 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3)
最終 3次元分類
クラス数: 4 / 平均メンバー数/クラス: 60000 / ソフトウェア - 名称: RELION (ver. 3) 詳細: The cryo-EM dataset was first classified for the subvolume of interest using signal-subtracted classification, and then classified globally to improve the overall quality of the reconstruction.