ジャーナル: Nature / 年: 2015 タイトル: The architecture of the spliceosomal U4/U6.U5 tri-snRNP. 著者: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-chen Bai / Christos G Savva / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai / 要旨: U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key ...U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3' stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome.
詳細: 400 mesh copper grid with thin carbon support, glow discharged in amylamine atmosphere
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 85 K / 装置: FEI VITROBOT MARK II / 手法: Blot for 2 s before plunging
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電子顕微鏡法
顕微鏡
FEI POLARA 300
温度
最低: 80 K / 最高: 90 K / 平均: 85 K
日付
2014年6月10日
撮影
カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 実像数: 2035 / 平均電子線量: 40 e/Å2 詳細: Every image is the average of sixteen frames recorded by the direct electron detector
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
倍率(補正後): 79096 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2 mm / 倍率(公称値): 59000
試料ステージ
試料ホルダーモデル: GATAN LIQUID NITROGEN
実験機器
モデル: Tecnai Polara / 画像提供: FEI Company
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画像解析
CTF補正
詳細: each particle
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 5.9 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: CTFFIND3, RELION 詳細: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction ...詳細: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction was performed using statistical movie processing in RELION. 使用した粒子像数: 179079