+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 7ksl | |||||||||||||||
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タイトル | Substrate-free human mitochondrial LONP1 | |||||||||||||||
要素 | Lon protease homolog, mitochondrial | |||||||||||||||
キーワード | HYDROLASE / AAA+ / ATPase / protease / mitochondrial / LONP1 / LON | |||||||||||||||
機能・相同性 | 機能・相同性情報 oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / negative regulation of insulin receptor signaling pathway / Mitochondrial protein degradation / 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 / single-stranded RNA binding / response to hypoxia / 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 | Homo sapiens (ヒト) | |||||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.5 Å | |||||||||||||||
データ登録者 | Shin, M. / Watson, E.R. / Song, A.S. / Mindrebo, J.T. / Novick, S.R. / Griffin, P. / Wiseman, R.L. / Lander, G.C. | |||||||||||||||
資金援助 | 米国, 4件
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引用 | ジャーナル: Nat Commun / 年: 2021 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 |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 7ksl.cif.gz | 418.1 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb7ksl.ent.gz | 335.2 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 7ksl.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 7ksl_validation.pdf.gz | 1.7 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 7ksl_full_validation.pdf.gz | 1.8 MB | 表示 | |
XML形式データ | 7ksl_validation.xml.gz | 74.4 KB | 表示 | |
CIF形式データ | 7ksl_validation.cif.gz | 112.6 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/ks/7ksl ftp://data.pdbj.org/pub/pdb/validation_reports/ks/7ksl | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質 | 分子量: 58498.098 Da / 分子数: 5 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: LONP1, PRSS15 / プラスミド: pET20b / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 株 (発現宿主): Rosetta 2(DE3)pLysS / 参照: UniProt: P36776, endopeptidase La #2: 化合物 | ChemComp-ADP / 研究の焦点であるリガンドがあるか | N | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
-試料調製
構成要素 | 名称: Substrate-free human mitochondrial LONP1 / タイプ: COMPLEX 詳細: Complexes consisting of homohexameric LONP1 protease from Homo sapiens Entity ID: #1 / 由来: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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分子量 | 値: 0.462 MDa / 実験値: NO | |||||||||||||||||||||||||||||||||||
由来(天然) | 生物種: Homo sapiens (ヒト) / 細胞内の位置: Matrix / Organelle: Mitochondria | |||||||||||||||||||||||||||||||||||
由来(組換発現) | 生物種: Escherichia coli BL21(DE3) (大腸菌) / 株: Rosetta 2(DE3)pLysS / プラスミド: pET20b | |||||||||||||||||||||||||||||||||||
緩衝液 | pH: 8 詳細: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated for 5 minutes prior to vitrification. | |||||||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 2.5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES / 詳細: This sample was monodisperse | |||||||||||||||||||||||||||||||||||
試料支持 | 詳細: EM 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. グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: UltrAuFoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 277 K 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. |
-電子顕微鏡撮影
実験機器 | モデル: Talos Arctica / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TALOS ARCTICA 詳細: Coma-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection. |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 200 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 36000 X / 倍率(補正後): 43478 X / 最大 デフォーカス(公称値): 1200 nm / 最小 デフォーカス(公称値): 800 nm / Calibrated defocus min: 500 nm / 最大 デフォーカス(補正後): 1500 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 90 K / 最低温度: 80 K / Residual tilt: 0.14 mradians |
撮影 | 平均露光時間: 11.4 sec. / 電子線照射量: 50 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 撮影したグリッド数: 1 / 実像数: 2912 詳細: Images were collected in counting mode at 10 frames per second. |
画像スキャン | サンプリングサイズ: 5 µm / 横: 3710 / 縦: 3838 / 動画フレーム数/画像: 114 / 利用したフレーム数/画像: 0-113 |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.15.2_3472: / 分類: 精密化 | |||||||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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CTF補正 | 詳細: CTF parameters were estimated with CTFFIND / タイプ: PHASE FLIPPING ONLY | |||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 564930 | |||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 83162 / クラス平均像の数: 2 / 対称性のタイプ: POINT | |||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 30 / プロトコル: AB INITIO MODEL / 空間: REAL / Target criteria: Correlation coefficient 詳細: Initial rigid body docking was done using UCSF Chimera. | |||||||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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