ジャーナル: Cell / 年: 2005 タイトル: The cryo-EM structure of a translation initiation complex from Escherichia coli. 著者: Gregory S Allen / Andrey Zavialov / Richard Gursky / Måns Ehrenberg / Joachim Frank / 要旨: The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex ...The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.
モード: BRIGHT FIELD / 倍率(公称値): 39000 X / 最大 デフォーカス(公称値): -3930 nm / 最小 デフォーカス(公称値): -930 nm / Cs: 2 mm
試料ホルダ
温度: 100 K / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 °
撮影
フィルム・検出器のモデル: KODAK SO-163 FILM
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解析
CTF補正
詳細: DEFOCUS GROUPS 0.93-3.93 UM
対称性
点対称性: C1 (非対称)
3次元再構成
手法: MULTI-REFERENCE / 解像度: 13.8 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 20283 / ピクセルサイズ(公称値): 2.82 Å / ピクセルサイズ(実測値): 2.82 Å 詳細: resolution 13.8 ANGSTROMS (FSC=0.5) and 8.6 ANGSTROMS (3sigma) 対称性のタイプ: POINT
原子モデル構築
プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: BEST FIT USING RSREF 詳細: REFINEMENT PROTOCOL--RIGID BODY FIT OF TRNA DETAILS--the tRNA coordinate files were fitted to the E. coli translation initiation complex map (EMBL-EMD 3525) in the following way- first the ...詳細: REFINEMENT PROTOCOL--RIGID BODY FIT OF TRNA DETAILS--the tRNA coordinate files were fitted to the E. coli translation initiation complex map (EMBL-EMD 3525) in the following way- first the cross-correlation of the respective 30S subunit maps were maximized and the resulting transformation matrix was computed. second the coordinates of the tRNA models were transformed according to the matrix previously computed.