ジャーナル: J Mol Biol / 年: 2008 タイトル: Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy. 著者: Jayati Sengupta / Jakob Nilsson / Richard Gursky / Morten Kjeldgaard / Poul Nissen / Joachim Frank / 要旨: In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum ...In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.
モード: BRIGHT FIELD / 倍率(公称値): 50000 X / 倍率(補正後): 49650 X / 最大 デフォーカス(公称値): 4500 nm / 最小 デフォーカス(公称値): 1500 nm
撮影
電子線照射量: 10 e/Å2 / 詳細: Kodak SO163 film
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解析
EMソフトウェア
ID
名称
カテゴリ
1
O
モデルフィッティング
2
SPIDER
3次元再構成
CTF補正
詳細: segregation in defocus groups and correction in volumes
対称性
点対称性: C1 (非対称)
3次元再構成
手法: SPIDER / 解像度: 12.6 Å / 解像度の算出法: FSC / 粒子像の数: 28242 / ピクセルサイズ(公称値): 2.82 Å 詳細: Single particle reconstruction, resolution estimated: FSC cut-off at 0.15 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: correlation coefficient 詳細: METHOD--manual REFINEMENT PROTOCOL--Fitted as rigid body. Current model was aligned to the helix A of the fitted eEF2 coordinates (PDB entry 3DNY) which is located next to the switch I ...詳細: METHOD--manual REFINEMENT PROTOCOL--Fitted as rigid body. Current model was aligned to the helix A of the fitted eEF2 coordinates (PDB entry 3DNY) which is located next to the switch I sequence. The coordinates for this entry are based on manual fitting of the coordinates into cryo-EM density map. Therefore, authors did not deposit structure factors.