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データを開く
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基本情報
登録情報 | ![]() | |||||||||
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タイトル | Escherichia coli paused disome complex (local refinement of leading 70S rotated PRE-1) | |||||||||
![]() | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Full map. | |||||||||
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![]() | ribosome / polysome / tranlsation / elongation / pausing / disome / collision / PURE system | |||||||||
生物種 | ![]() ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.45 Å | |||||||||
![]() | Fluegel T / Schacherl M | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM. 著者: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén ...著者: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén Schlippe / Christian M T Spahn / ![]() 要旨: Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex ...Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts. | |||||||||
履歴 |
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構造の表示
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ダウンロードとリンク
-EMDBアーカイブ
マップデータ | ![]() | 186.4 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 25 KB 25 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 15.4 KB | 表示 | ![]() |
画像 | ![]() | 53.1 KB | ||
マスクデータ | ![]() | 391 MB | ![]() | |
Filedesc metadata | ![]() | 5 KB | ||
その他 | ![]() ![]() | 362.3 MB 362.3 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
EMDBのページ | ![]() ![]() |
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マップ
ファイル | ![]() | ||||||||||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Full map. | ||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.59 Å | ||||||||||||||||||||
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対称性 | 空間群: 1 | ||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-マスク #1
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投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Cryo-EM reconstruction of the E. coli disome complex...
ファイル | emd_19096_half_map_1.map | ||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Half map A. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Cryo-EM reconstruction of the E. coli disome complex...
ファイル | emd_19096_half_map_2.map | ||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Half map B. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
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試料の構成要素
-全体 : Escherichia coli paused disome complex
全体 | 名称: Escherichia coli paused disome complex |
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要素 |
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-超分子 #1: Escherichia coli paused disome complex
超分子 | 名称: Escherichia coli paused disome complex / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#33, #35-#60 詳細: Model of leading ribosome, focused refinement on leading ribosome applied on disome map |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 理論値: 2.270379 MDa |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
緩衝液 | pH: 7.6 |
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グリッド | モデル: Quantifoil R2/2 / 材質: COPPER / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 75 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher ...詳細: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher Scientific) after a wait time of 40 s at 4 degrees Celcius.. |
詳細 | In vitro translation reactions were performed in the PURE translation system using the PURExpress delta ribosome kit (NEB, #E3313S). Translation reactions were supplemented with 0.8 U/uL RNAsin Plus RNase Inhibitor (Promega, N261B). SolA, factor mix, and RNAsin Plus were combined on ice, followed by a preincubation at 37 degrees Celcius for 2 min, and added directly to polysomes (700 nM final concentration) that had been preincubated at 37 degrees Celsius for 2 min. After 1 min reaction time, 4 uL of the reaction mixture were withdrawn for plunge freezing. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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温度 | 最低: 80.0 K / 最高: 82.0 K |
ソフトウェア | 名称: EPU (ver. 2.8.1) |
撮影 | フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 撮影したグリッド数: 1 / 実像数: 8982 / 平均露光時間: 1.13 sec. / 平均電子線量: 45.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 50.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 0.5 µm / 倍率(公称値): 81000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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画像解析
-原子モデル構築 1
ソフトウェア | 名称: ![]() |
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