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基本情報
登録情報 | データベース: EMDB / ID: EMD-5799 | |||||||||
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タイトル | Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State | |||||||||
![]() | Reconstruction of a pre-translocation ribosome | |||||||||
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![]() | protein structure / translation / EF-G / electron cryo-microscopy / single particle analysis | |||||||||
機能・相同性 | ![]() stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis ...stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / ribosomal large subunit assembly / transcription antitermination / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / molecular adaptor activity / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm 類似検索 - 分子機能 | |||||||||
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: 7.6 Å | |||||||||
![]() | Brilot AF / Korostelev AA / Ermolenko DN / Grigorieff N | |||||||||
![]() | ![]() タイトル: Structure of the ribosome with elongation factor G trapped in the pretranslocation state. 著者: Axel F Brilot / Andrei A Korostelev / Dmitri N Ermolenko / Nikolaus Grigorieff / ![]() 要旨: During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by ...During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state. | |||||||||
履歴 |
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ヘッダ (付随情報) | ![]() ![]() | 16 KB 16 KB | 表示 表示 | ![]() |
画像 | ![]() ![]() | 194.2 KB 211.6 KB | ||
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「今月の分子」の関連する項目 |
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注釈 | Reconstruction of a pre-translocation ribosome | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.04 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
-全体 : Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
全体 | 名称: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA |
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要素 |
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-超分子 #1000: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
超分子 | 名称: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA タイプ: sample / ID: 1000 / 詳細: Sample was monodisperse. / Number unique components: 4 |
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分子量 | 理論値: 3 MDa |
-超分子 #1: 70S ribosome
超分子 | 名称: 70S ribosome / タイプ: complex / ID: 1 / 組換発現: No / データベース: NCBI / Ribosome-details: ribosome-prokaryote: ALL |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 理論値: 3 MDa |
-分子 #1: Transfer RNA
分子 | 名称: Transfer RNA / タイプ: rna / ID: 1 / Name.synonym: tRNA / 分類: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 実験値: 25 KDa |
-分子 #2: Messenger RNA
分子 | 名称: Messenger RNA / タイプ: rna / ID: 2 / Name.synonym: mRNA / 分類: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes |
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由来(天然) | 生物種: synthetic construct (人工物) |
分子量 | 理論値: 12 KDa |
配列 | 文字列: GGCAAGGAGG UAAAAAUGUU UAAACGUAAA UCUACU |
-分子 #3: Viomycin
分子 | 名称: Viomycin / タイプ: ligand / ID: 3 / Name.synonym: Vio / コピー数: 1 / 組換発現: No / データベース: NCBI |
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由来(天然) | 生物種: unidentified (未定義) |
分子量 | 理論値: 1 KDa |
Chemical component information | ![]()
ChemComp-PRD_000226: |
-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
濃度 | 0.4 mg/mL |
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緩衝液 | pH: 7.6 詳細: 10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acid |
グリッド | 詳細: C-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 95 % / 装置: FEI VITROBOT MARK II 手法: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side ...手法: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side port, blotted for 7 seconds with a positional offset of 2, and plunged into liquid ethane. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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アライメント法 | Legacy - 非点収差: Automatically corrected using FEI software |
日付 | 2012年11月2日 |
撮影 | カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON I (4k x 4k) デジタル化 - サンプリング間隔: 14.0 µm / 実像数: 13341 / 平均電子線量: 30 e/Å2 / ビット/ピクセル: 16 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 倍率(補正後): 134615 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 0.01 mm / 最大 デフォーカス(公称値): 6.95 µm / 最小 デフォーカス(公称値): 1.15 µm / 倍率(公称値): 133333 |
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
詳細 | Refinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details. |
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CTF補正 | 詳細: CTFFIND3, FREALIGN per micrograph |
最終 再構成 | アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 7.6 Å / 解像度の算出法: OTHER ソフトウェア - 名称: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3 詳細: Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details. 使用した粒子像数: 85115 |
-原子モデル構築 1
初期モデル | PDB ID: ![]() 4gd1 |
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ソフトウェア | 名称: Chimera, CNS |
詳細 | Chain Y not used in refinement. See primary citation Supplementary Information for details. |
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 |
得られたモデル | ![]() PDB-4v7c: |
-原子モデル構築 2
初期モデル | PDB ID: ![]() 3r8s |
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ソフトウェア | 名称: Chimera, CNS |
詳細 | See primary citation Supplementary Information for details. |
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 |
得られたモデル | ![]() PDB-4v7c: |
-原子モデル構築 3
初期モデル | PDB ID: |
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ソフトウェア | 名称: Chimera, CNS |
詳細 | Used to rebuild the L11 stalk. |
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 |
得られたモデル | ![]() PDB-4v7c: |
-原子モデル構築 4
初期モデル | PDB ID: |
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ソフトウェア | 名称: Chimera, CNS |
詳細 | Used to build homology model for L1. |
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 |
得られたモデル | ![]() PDB-4v7c: |