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- EMDB-2978: Time-resolved Cryo Electron Microscopy of ribosome subunit association -
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基本情報
登録情報 | データベース: EMDB / ID: EMD-2978 | |||||||||
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タイトル | Time-resolved Cryo Electron Microscopy of ribosome subunit association | |||||||||
![]() | Reconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation | |||||||||
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![]() | time-resolved / cryo-EM / mixing-spraying / ribosome subunit association / structural dynamics | |||||||||
機能・相同性 | ![]() Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / protein catabolic process at postsynapse / amyloid precursor protein biosynthetic process / positive regulation of coagulation / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process ...Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / protein catabolic process at postsynapse / amyloid precursor protein biosynthetic process / positive regulation of coagulation / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process / positive regulation of endopeptidase activity / Noncanonical activation of NOTCH3 / sequestering of calcium ion / Notch receptor processing / choline transport / central nervous system myelination / synaptic vesicle targeting / membrane protein intracellular domain proteolysis / negative regulation of axonogenesis / regulation of resting membrane potential / T cell activation involved in immune response / NOTCH4 Activation and Transmission of Signal to the Nucleus / skin morphogenesis / growth factor receptor binding / dorsal/ventral neural tube patterning / regulation of synaptic vesicle cycle / neural retina development / L-glutamate import across plasma membrane / myeloid dendritic cell differentiation / Regulated proteolysis of p75NTR / regulation of phosphorylation / brain morphogenesis / endoplasmic reticulum calcium ion homeostasis / nuclear outer membrane / glutamate receptor signaling pathway / locomotion / smooth endoplasmic reticulum calcium ion homeostasis / amyloid precursor protein metabolic process / regulation of canonical Wnt signaling pathway / astrocyte activation involved in immune response / aggresome / regulation of long-term synaptic potentiation / skeletal system morphogenesis / embryonic limb morphogenesis / cell fate specification / ciliary rootlet / myeloid cell homeostasis / regulation of postsynapse organization / azurophil granule membrane / regulation of neuron projection development / G protein-coupled dopamine receptor signaling pathway / 加水分解酵素; プロテアーゼ; ペプチド結合加水分解酵素; アスパラギン酸プロテアーゼ / positive regulation of amyloid fibril formation / adult behavior / positive regulation of dendritic spine development / positive regulation of receptor recycling / mitochondrial transport / positive regulation of catalytic activity / blood vessel development / heart looping / protein glycosylation / cerebral cortex cell migration / amyloid precursor protein catabolic process / amyloid-beta formation / negative regulation of apoptotic signaling pathway / membrane protein ectodomain proteolysis / autophagosome assembly / EPH-ephrin mediated repulsion of cells / smooth endoplasmic reticulum / neuron development / hematopoietic progenitor cell differentiation / somitogenesis / T cell proliferation / Nuclear signaling by ERBB4 / rough endoplasmic reticulum / viral release from host cell by cytolysis / Notch signaling pathway / regulation of synaptic transmission, glutamatergic / NOTCH2 Activation and Transmission of Signal to the Nucleus / neuron projection maintenance / cellular response to calcium ion / positive regulation of glycolytic process / Degradation of the extracellular matrix / negative regulation of ubiquitin-dependent protein catabolic process / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / peptidoglycan catabolic process / cerebellum development / post-embryonic development / thymus development / negative regulation of protein phosphorylation / dendritic shaft / epithelial cell proliferation / NOTCH3 Activation and Transmission of Signal to the Nucleus / PDZ domain binding / astrocyte activation / apoptotic signaling pathway / synapse organization / neuron migration / calcium channel activity 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: 11.6 Å | |||||||||
![]() | Chen B / Kaledhonkar S / Sun M / Shen B / Lu Z / Barnard D / Lu T / Gonzalez Jr R / Frank J | |||||||||
![]() | ![]() タイトル: Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. 著者: Bo Chen / Sandip Kaledhonkar / Ming Sun / Bingxin Shen / Zonghuan Lu / David Barnard / Toh-Ming Lu / Ruben L Gonzalez / Joachim Frank / ![]() 要旨: Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, ...Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method. | |||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
添付画像 |
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画像 | ![]() | 281.5 KB | ||
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文書・要旨 | ![]() | 226 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 225.2 KB | 表示 | |
XML形式データ | ![]() | 5.4 KB | 表示 | |
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「今月の分子」の関連する項目 |
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注釈 | Reconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 2.2451 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 : E. Coli 70S Ribosome
全体 | 名称: E. Coli 70S Ribosome |
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要素 |
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-超分子 #1000: E. Coli 70S Ribosome
超分子 | 名称: E. Coli 70S Ribosome / タイプ: sample / ID: 1000 / Number unique components: 1 |
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-超分子 #1: 70S ribosome
超分子 | 名称: 70S ribosome / タイプ: complex / ID: 1 / 組換発現: No / Ribosome-details: ribosome-prokaryote: LSU 50S, SSU 30S |
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由来(天然) | 生物種: ![]() ![]() |
-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 詳細: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2 |
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グリッド | 詳細: Quantifoil R2/2 300 mesh copper grid with thin carbon sipport |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 80 % / チャンバー内温度: 80 K / 装置: OTHER 詳細: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled ...詳細: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled plunging device was purchased from Dr. Howard White (Eastern Virginia Medical School, VA). Timed resolved state: Vitrified after spraying |
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電子顕微鏡法
顕微鏡 | FEI TECNAI F20 |
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温度 | 平均: 80 K |
詳細 | Low dose, Data was collected over two years time |
日付 | 2013年9月13日 |
撮影 | カテゴリ: CCD フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) 実像数: 3402 / 平均電子線量: 17 e/Å2 |
電子線 | 加速電圧: 200 kV / 電子線源: ![]() |
電子光学系 | 倍率(補正後): 66318 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2 mm / 最大 デフォーカス(公称値): 4.0 µm / 最小 デフォーカス(公称値): 2.0 µm / 倍率(公称値): 50000 |
試料ステージ | 試料ホルダー: CT 3500 / 試料ホルダーモデル: GATAN LIQUID NITROGEN |
実験機器 | ![]() モデル: Tecnai F20 / 画像提供: FEI Company |
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画像解析
詳細 | The partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION |
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CTF補正 | 詳細: each Micrograph |
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: 11.6 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Arachnid, RELION, SPIDER / 使用した粒子像数: 11129 |