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- EMDB-23320: WalkerB mutant human mitochondrial LONP1 bound to endogenous substrate -
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データを開く
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基本情報
登録情報 | データベース: EMDB / ID: EMD-23320 | |||||||||||||||
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タイトル | WalkerB mutant human mitochondrial LONP1 bound to endogenous substrate | |||||||||||||||
![]() | Final EM map of substrate-bound, WalkerB mutant human mitochondrial LONP1 | |||||||||||||||
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機能・相同性 | ![]() oxidation-dependent protein catabolic process / response to aluminum ion / PH domain binding / endopeptidase La / mitochondrial protein catabolic process / mitochondrial DNA metabolic process / G-quadruplex DNA binding / : / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins ...oxidation-dependent protein catabolic process / response to aluminum ion / PH domain binding / endopeptidase La / mitochondrial protein catabolic process / mitochondrial DNA metabolic process / G-quadruplex DNA binding / : / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / Mitochondrial unfolded protein response (UPRmt) / chaperone-mediated protein complex assembly / response to hormone / DNA polymerase binding / Mitochondrial protein degradation / negative regulation of insulin receptor signaling pathway / proteolysis involved in protein catabolic process / mitochondrion organization / protein catabolic process / ADP binding / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / response to hypoxia / single-stranded RNA binding / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / membrane / cytosol 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: 4.8 Å | |||||||||||||||
![]() | Shin M / Watson ER / Song AS / Novick SR / Griffin P / Wiseman RL / Lander GC | |||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structures of the human LONP1 protease reveal regulatory steps involved in protease activation. 著者: Mia Shin / Edmond R Watson / Albert S Song / Jeffrey T Mindrebo / Scott J Novick / Patrick R Griffin / R Luke Wiseman / Gabriel C Lander / ![]() 要旨: The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease. | |||||||||||||||
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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マップデータ | ![]() | 136.6 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 23.2 KB 23.2 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 15.5 KB | 表示 | ![]() |
画像 | ![]() | 91.2 KB | ||
その他 | ![]() ![]() | 134.4 MB 134.4 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-検証レポート
文書・要旨 | ![]() | 604.6 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 604.2 KB | 表示 | |
XML形式データ | ![]() | 19.9 KB | 表示 | |
CIF形式データ | ![]() | 25.7 KB | 表示 | |
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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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Final EM map of substrate-bound, WalkerB mutant human mitochondrial LONP1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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: 1.15 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
-ハーフマップ: Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1
ファイル | emd_23320_half_map_1.map | ||||||||||||
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注釈 | Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1
ファイル | emd_23320_half_map_2.map | ||||||||||||
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注釈 | Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1 | ||||||||||||
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 : WalkerB mutant Human mitochondrial LONP1
全体 | 名称: WalkerB mutant Human mitochondrial LONP1 |
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要素 |
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-超分子 #1: WalkerB mutant Human mitochondrial LONP1
超分子 | 名称: WalkerB mutant Human mitochondrial LONP1 / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: Complexes consisting of homohexameric LONP1 protease with an E591A mutation from Homo sapiens bound to endogenous co-purified substrate |
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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() ![]() |
分子量 | 理論値: 462 KDa |
-分子 #1: WalkerB mutant Human mitochondrial LONP1
分子 | 名称: WalkerB mutant Human mitochondrial LONP1 / タイプ: protein_or_peptide / ID: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: AIEEKFRERL KELVVPKHVM DVVDEELSKL GLLDNHSSEF NVTRNYLDWL TSIPWGKYSN ENLDLARAQA VLEEDHYGME DVKKRILEFI AVSQLRGSTQ GKILCFYGPP GVGKTSIARS IARALNREYF RFSVGGMTDV AEIKGHRRTY VGALKKTKTE NPLILIDAVD ...文字列: AIEEKFRERL KELVVPKHVM DVVDEELSKL GLLDNHSSEF NVTRNYLDWL TSIPWGKYSN ENLDLARAQA VLEEDHYGME DVKKRILEFI AVSQLRGSTQ GKILCFYGPP GVGKTSIARS IARALNREYF RFSVGGMTDV AEIKGHRRTY VGALKKTKTE NPLILIDAVD KIGRGYQGDP SSALLELLDP EQNANFYLDV PVDLSKVLFI CTANVTDTIP EPLRDRMEMI NVSGYVAQEK LAIAERYLVP QARALCGLDE SKAKLSSDVL TLLIKQYCRE SGVRNLQKQV EKVLRKSAYK IVSGEAESVE VTPENLQDFV GKPVFTVERM VTPPGVVMGL AWTAMGGSTL FVETSLRRPG DKDGSLEVTG QLGEVMKESA RIAYTFARAF LMQHAPANDY LVTSHIHLHV PEGATPKDGP SAGCTIVTAL LSLAMGRPVR QNLAMTGEVS LTGKILPVGG IKEKTIAAKR AGVTCIVLPA ENKKDFYDLA AFITEGLEVH FVEHYREIFD IAF |
-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 | 2.5 mg/mL | |||||||||||||||||||||
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緩衝液 | pH: 8 構成要素:
詳細: Solutions were made fresh from concentrated stocks and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated with fresh ATP for 5 ...詳細: Solutions were made fresh from concentrated stocks and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated with fresh ATP for 5 minutes prior to vitrification. | |||||||||||||||||||||
グリッド | モデル: UltrAuFoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: GOLD / 支持フィルム - トポロジー: HOLEY / 支持フィルム - Film thickness: 50.0 nm / 前処理 - タイプ: PLASMA CLEANING / 前処理 - 雰囲気: OTHER 詳細: Grids were plasma cleaned prior to sample application for 7 seconds using a Solarus plasma cleaner (Gatan, Inc.) with a 75% nitrogen, 25% oxygen atmosphere at 15W. | |||||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 277 K / 装置: HOMEMADE PLUNGER 詳細: 4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen.. | |||||||||||||||||||||
詳細 | This sample was monodisperse. |
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電子顕微鏡法
顕微鏡 | FEI TALOS ARCTICA |
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温度 | 最低: 80.0 K / 最高: 90.0 K |
アライメント法 | Coma free - Residual tilt: 0.14 mrad |
詳細 | Coma-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection. |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - サイズ - 横: 3710 pixel / デジタル化 - サイズ - 縦: 3838 pixel / デジタル化 - サンプリング間隔: 5.0 µm / デジタル化 - 画像ごとのフレーム数: 0-58 / 撮影したグリッド数: 1 / 実像数: 2415 / 平均露光時間: 11.8 sec. / 平均電子線量: 50.0 e/Å2 詳細: Images were collected in counting mode at 5 frames per second. |
電子線 | 加速電圧: 200 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 70.0 µm / 最大 デフォーカス(補正後): 1.5 µm / 最小 デフォーカス(補正後): 0.5 µm / 倍率(補正後): 43478 / 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): 1.2 µm / 最小 デフォーカス(公称値): 0.8 µm / 倍率(公称値): 36000 |
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: Talos Arctica / 画像提供: FEI Company |