ジャーナル: EMBO J / 年: 2007 タイトル: Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. 著者: Derek J Taylor / Jakob Nilsson / A Rod Merrill / Gregers Rom Andersen / Poul Nissen / Joachim Frank / 要旨: On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 ...On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.
モード: BRIGHT FIELD / 倍率(公称値): 39000 X / 倍率(補正後): 37642 X / 最大 デフォーカス(公称値): 3700 nm / 最小 デフォーカス(公称値): 800 nm / Cs: 2.26 mm
試料ホルダ
温度: 93 K / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 °
撮影
電子線照射量: 25 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
放射
プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M
放射波長
相対比: 1
-
解析
EMソフトウェア
ID
名称
カテゴリ
詳細
1
RSRef
モデルフィッティング
TNTimplementationofRSRef
2
SPIDER
3次元再構成
CTF補正
詳細: CTF correction of 3D-maps by Wiener filtration
対称性
点対称性: C1 (非対称)
3次元再構成
手法: 3D projection matching, conjugate gradients with regularization 解像度: 11.7 Å / 粒子像の数: 70510 / ピクセルサイズ(公称値): 1.86 Å / ピクセルサイズ(実測値): 1.86 Å / 倍率補正: TMV / 詳細: SPIDER package / 対称性のタイプ: POINT
原子モデル構築
B value: 15 / プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: Maximization of correlation-coefficient 詳細: METHOD--Real-space refinement using rigid bodies REFINEMENT PROTOCOL--TNT implementation of RSRef