ジャーナル: Nature / 年: 2006 タイトル: Structure of eEF3 and the mechanism of transfer RNA release from the E-site. 著者: Christian B F Andersen / Thomas Becker / Michael Blau / Monika Anand / Mario Halic / Bharvi Balar / Thorsten Mielke / Thomas Boesen / Jan Skov Pedersen / Christian M T Spahn / Terri Goss ...著者: Christian B F Andersen / Thomas Becker / Michael Blau / Monika Anand / Mario Halic / Bharvi Balar / Thorsten Mielke / Thomas Boesen / Jan Skov Pedersen / Christian M T Spahn / Terri Goss Kinzy / Gregers R Andersen / Roland Beckmann / 要旨: Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of ...Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.
全体 : 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
全体
名称: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
要素
試料: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
複合体: programmed 80S ribosome
タンパク質・ペプチド: elongation factor 3
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超分子 #1000: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
超分子
名称: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae / タイプ: sample / ID: 1000 詳細: The emerging signal sequence of the ribosome nascent chain (RNC) was saturated using purified trimeric Sec61 complex in order to prevent biased orientation of particles 集合状態: One ribosome binds to one molecule of eEF3 / Number unique components: 2
分子量
理論値: 4.3 MDa
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超分子 #1: programmed 80S ribosome
超分子
名称: programmed 80S ribosome / タイプ: complex / ID: 1 / Name.synonym: RNC / 詳細: programmed 80S ribosome with a P-site tRNA / Ribosome-details: ribosome-eukaryote: ALL
分子量
実験値: 4.2 MDa / 理論値: 4.2 MDa
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分子 #1: elongation factor 3
分子
名称: elongation factor 3 / タイプ: protein_or_peptide / ID: 1 / Name.synonym: eEF3 / 詳細: eEF3 with a C-terminal His-Tag and a factor / コピー数: 1 / 集合状態: Monomer / 組換発現: Yes
pH: 7.5 詳細: 20 mM HEPES, pH 7.5, 10 mM Mg(OAc)2, 150 mM KOAc 1 mM DTT, 0.05% Nikkol, 125 mM Sucrose,0.01 mg ml-1 Cycloheximide, 0.5 mM AMP-PNP, 0.1 mM Neomycin, 0.3 % DesoxyBigChaps
染色
タイプ: NEGATIVE / 詳細: Cryo-EM, no staining
グリッド
詳細: Quantifoil grids 200 mesh R2/4
凍結
凍結剤: ETHANE / チャンバー内湿度: 95 % / 装置: OTHER / 詳細: Vitrification instrument: Vitrobot 手法: Blot for 10 seconds before plunging, use 2 layers of filter paper
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電子顕微鏡法
顕微鏡
FEI TECNAI F30
温度
最低: 95 K / 平均: 95 K
アライメント法
Legacy - 非点収差: objective lens astigmatism was corrected at 100,000 time