+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-19059 | |||||||||
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タイトル | Escherichia coli paused disome complex (Rotated disome interface class 2) | |||||||||
マップデータ | Cryo-EM reconstruction of the E. coli disome complex (rotated disome interface class 2). Full map. | |||||||||
試料 |
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キーワード | polysome / translation / pausing / disome / RIBOSOME | |||||||||
機能・相同性 | 機能・相同性情報 RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation ...RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / DNA endonuclease activity / transcription antitermination / translational initiation / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / ribosomal large subunit assembly / small ribosomal subunit / transferase activity / large ribosomal subunit rRNA binding / 5S rRNA binding / cytosolic small ribosomal subunit / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / single-stranded RNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | Escherichia coli (大腸菌) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 5.32 Å | |||||||||
データ登録者 | Fluegel T / Schacherl M | |||||||||
資金援助 | ドイツ, 1件
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引用 | ジャーナル: Nat Commun / 年: 2024 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
添付画像 |
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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 | emd_19059.map.gz | 327.1 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-19059-v30.xml emd-19059.xml | 66.2 KB 66.2 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_19059_fsc.xml | 15.5 KB | 表示 | FSCデータファイル |
画像 | emd_19059.png | 64.4 KB | ||
マスクデータ | emd_19059_msk_1.map | 391 MB | マスクマップ | |
Filedesc metadata | emd-19059.cif.gz | 14.6 KB | ||
その他 | emd_19059_half_map_1.map.gz emd_19059_half_map_2.map.gz | 362.3 MB 362.3 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-19059 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-19059 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_19059_validation.pdf.gz | 1.3 MB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_19059_full_validation.pdf.gz | 1.3 MB | 表示 | |
XML形式データ | emd_19059_validation.xml.gz | 25.1 KB | 表示 | |
CIF形式データ | emd_19059_validation.cif.gz | 32.8 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-19059 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-19059 | HTTPS FTP |
-関連構造データ
関連構造データ | 8rctMC 8pegC 8pklC 8r3vC 8rclC 8rcmC 8rcsC C: 同じ文献を引用 (文献) M: このマップから作成された原子モデル |
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類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_19059.map.gz / 形式: CCP4 / 大きさ: 391 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (rotated disome interface class 2). Full map. | ||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.59 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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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
ファイル | emd_19059_msk_1.map | ||||||||||||
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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_19059_half_map_1.map | ||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (rotated disome interface class 2). Half map B. | ||||||||||||
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_19059_half_map_2.map | ||||||||||||
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注釈 | Cryo-EM reconstruction of the E. coli disome complex (rotated disome interface class 2). Half map A. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-試料の構成要素
+全体 : Escherichia coli paused disome complex
+超分子 #1: Escherichia coli paused disome complex
+分子 #1: 50S ribosomal protein L28
+分子 #2: 50S ribosomal protein L30
+分子 #3: Large ribosomal subunit protein bL31A
+分子 #4: 50S ribosomal protein L35
+分子 #8: Small ribosomal subunit protein uS2
+分子 #9: Small ribosomal subunit protein uS3
+分子 #10: Small ribosomal subunit protein uS4
+分子 #11: Small ribosomal subunit protein uS5
+分子 #12: 30S ribosomal protein S6
+分子 #13: 30S ribosomal protein S7
+分子 #14: Small ribosomal subunit protein uS8
+分子 #15: Small ribosomal subunit protein uS9
+分子 #16: 30S ribosomal protein S10
+分子 #17: Small ribosomal subunit protein uS11
+分子 #18: Small ribosomal subunit protein uS12
+分子 #19: Small ribosomal subunit protein uS13
+分子 #20: Small ribosomal subunit protein uS14
+分子 #21: 30S ribosomal protein S15
+分子 #22: 30S ribosomal protein S16
+分子 #23: Small ribosomal subunit protein uS17
+分子 #24: Small ribosomal subunit protein bS18
+分子 #25: Small ribosomal subunit protein uS19
+分子 #26: Small ribosomal subunit protein bS20
+分子 #27: 30S ribosomal protein S21
+分子 #33: 30S ribosomal protein S1
+分子 #34: Large ribosomal subunit protein uL1
+分子 #35: 50S ribosomal protein L2
+分子 #36: 50S ribosomal protein L5
+分子 #37: 50S ribosomal protein L33
+分子 #38: 50S ribosomal protein L16
+分子 #39: 50S ribosomal protein L9
+分子 #40: 50S ribosomal protein L34
+分子 #41: 50S ribosomal protein L15
+分子 #42: Nascent chain
+分子 #43: 50S ribosomal protein L18
+分子 #44: 50S ribosomal protein L27
+分子 #5: 23S ribosomal RNA
+分子 #6: 5S ribosomal RNA
+分子 #7: 16S ribosomal RNA
+分子 #28: messenger RNA
+分子 #29: tRNA-Trp (P/E-site)
+分子 #30: tRNA-Phe (P/E-site)
+分子 #31: tRNA-Ala (A/P-site)
+分子 #32: tRNA-Val (A/P-site)
+分子 #45: ZINC ION
+分子 #46: 1,4-DIAMINOBUTANE
+分子 #47: SPERMIDINE
+分子 #48: MAGNESIUM ION
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-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 / 前処理 - 時間: 20 sec. / 前処理 - 雰囲気: AIR / 前処理 - 気圧: 0.037 kPa 詳細: Operated at 15 mA, easiGlow Discharge Cleaning system (PELCO) |
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. |
-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: FIELD EMISSION GUN |
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 |
+画像解析
-原子モデル構築 1
初期モデル | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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ソフトウェア | 名称: Coot (ver. 0.9.6) |
詳細 | The atomic model of the E. coli 70S ribosome (PDB ID: 7N1P) was initially docked into the postprocessed density maps with UCSF Chimera and manually adjusted in Coot. All models were refined over multiple rounds using the module phenix.real_space_refine in PHENIX and interactive model building and refinement in Coot, using libG restraints for the RNAs. |
精密化 | 空間: REAL / プロトコル: RIGID BODY FIT 当てはまり具合の基準: Cross-correlation coefficient |
得られたモデル | PDB-8rct: |