ジャーナル: Mol Cell / 年: 2015 タイトル: Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution. 著者: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao ...著者: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao Wang / Justyna Wojdyla / Lanjuan Li / Jiawei Wang / Meitian Wang / Genhong Cheng / Hong-Wei Wang / Yingfang Liu / 要旨: Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the ...Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
pH: 7.8 / 詳細: 25 mM HEPES 200 mM NaCl and 1 mM EDTA
グリッド
詳細: Quantifoil Cu R2/1
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV 手法: The grids were blotted for 6.5 s with 100% humidity and flash frozen in liquid ethane cooled at liquid nitrogen temperature using an FEI Vitrobot.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
日付
2014年10月2日
撮影
カテゴリ: CCD / フィルム・検出器のモデル: GATAN K2 (4k x 4k) / 平均電子線量: 50 e/Å2 詳細: Each image was fractionated into 32 sub-frames, with 0.25 s exposure time per frame. All dose-fractionated cryo-EM images were recorded using a semi-automated low-dose acquisition program UCSF-Image4
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 倍率(公称値): 22500
試料ステージ
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
最終 再構成
解像度のタイプ: BY AUTHOR / 解像度: 6.8 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: EMAN2, RELION / 使用した粒子像数: 67066