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基本情報
登録情報 | データベース: PDB / ID: 9nca | ||||||||||||
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タイトル | MicroED structure of microcrystals soaked with a mixture of E-64, E-64C, and E-64D | ||||||||||||
![]() | Papain | ||||||||||||
![]() | HYDROLASE/INHIBITOR / Inhibitor / Complex / MicroED / Cocktail / HYDROLASE-INHIBITOR complex | ||||||||||||
機能・相同性 | ![]() | ||||||||||||
生物種 | ![]() ![]() | ||||||||||||
手法 | 電子線結晶学 / ![]() | ||||||||||||
![]() | Vlahakis, N. / Rodriguez, J.A. | ||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Combining MicroED and native mass spectrometry for structural discovery of enzyme-small molecule complexes. 著者: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew P Agdanowski / Samuel Johnson / Jacob A Summers / Lian M C Jacobs / Catherine Keyser / Phoebe Russell / Samuel L Rose / Julien ...著者: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew P Agdanowski / Samuel Johnson / Jacob A Summers / Lian M C Jacobs / Catherine Keyser / Phoebe Russell / Samuel L Rose / Julien Orlans / Nima Adhami / Yu Chen / Michael R Sawaya / Shibom Basu / Daniele de Sanctis / Yu Chen / Soichi Wakatsuki / Hosea M Nelson / Joseph A Loo / Yi Tang / Jose A Rodriguez / ![]() ![]() 要旨: With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection ...With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach, which we term electron diffraction with native mass spectrometry (ED-MS), allows assignment of protein target structures bound to ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors and crude biosynthetic reactions. This extends to libraries of printed ligands dispensed directly onto TEM grids for later soaking with microcrystal slurries, and complexes with noncovalent ligands. ED-MS resolves structures of the natural product, epoxide-based cysteine protease inhibitor E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. It further identifies papain binding to its preferred natural products, by showing that two analogs of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analog from crude biosynthetic reactions, without purification. ED-MS also resolves binding of the CTX-M-14 β-lactamase, a target of active drug development, to the non-β-lactam inhibitor, avibactam, alone or in a cocktail of unrelated compounds. These results illustrate the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets, promising utility for drug discovery. #1: ![]() タイトル: Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes. 著者: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew Agdanowski / Samuel Johnson / Jacob A Summers / Catherine Keyser / Phoebe Russell / Samuel Rose / Julien Orlans / Nima Adhami / Yu ...著者: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew Agdanowski / Samuel Johnson / Jacob A Summers / Catherine Keyser / Phoebe Russell / Samuel Rose / Julien Orlans / Nima Adhami / Yu Chen / Michael R Sawaya / Shibom Basu / Daniele de Sanctis / Soichi Wakatsuki / Hosea M Nelson / Joseph A Loo / Yi Tang / Jose A Rodriguez / ![]() ![]() 要旨: With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection ...With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets. | ||||||||||||
履歴 |
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構造の表示
構造ビューア | 分子: ![]() ![]() |
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ダウンロードとリンク
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PDBx/mmCIF形式 | ![]() | 57.7 KB | 表示 | ![]() |
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PDB形式 | ![]() | 39.2 KB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
文書・要旨 | ![]() | 533.6 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 537.3 KB | 表示 | |
XML形式データ | ![]() | 11.5 KB | 表示 | |
CIF形式データ | ![]() | 14.4 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 9nbpC ![]() 9nbqC ![]() 9nc1C ![]() 9nccC ![]() 9oqeC ![]() 9or3C ![]() 9or7C ![]() 9orbC ![]() 9orgC ![]() 9orhC ![]() 9orlC ![]() 9orsC ![]() 9orvC ![]() 9orwC ![]() 9orxC ![]() 9oryC ![]() 9orzC ![]() 9os0C ![]() 9os1C ![]() 9os8C C: 同じ文献を引用 ( |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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リンク
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集合体
登録構造単位 | ![]()
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単位格子 |
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要素
#1: タンパク質 | 分子量: 23452.301 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() |
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#2: 化合物 | ChemComp-E64 / |
#3: 化合物 | ChemComp-E6C / |
#4: 水 | ChemComp-HOH / |
研究の焦点であるリガンドがあるか | Y |
Has protein modification | Y |
-実験情報
-実験
実験 | 手法: 電子線結晶学 |
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EM実験 | 試料の集合状態: 3D ARRAY / 3次元再構成法: 電子線結晶学 |
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試料調製
構成要素 | 名称: Papain-E-64/Papain-E-64C complex / タイプ: COMPLEX / Entity ID: #1 / 由来: NATURAL |
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由来(天然) | 生物種: ![]() ![]() |
緩衝液 | pH: 7 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 凍結剤: ETHANE |
-データ収集
実験機器 | ![]() モデル: Tecnai F20 / 画像提供: FEI Company |
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顕微鏡 | モデル: TFS TALOS F200C |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: DIFFRACTION / 最大 デフォーカス(公称値): 10000 nm / 最小 デフォーカス(公称値): 10000 nm / C2レンズ絞り径: 70 µm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER 最高温度: 100 K / 最低温度: 100 K |
撮影 | 電子線照射量: 0.045 e/Å2 フィルム・検出器のモデル: DIRECT ELECTRON APOLLO (4k x 4k) |
EM回折 シェル | 解像度: 2.5→2.6 Å / フーリエ空間範囲: 76.9 % / 多重度: 8.2 / 構造因子数: 638 / 位相残差: 30.57 ° |
EM回折 統計 | フーリエ空間範囲: 81.5 % / 再高解像度: 2.5 Å / 測定した強度の数: 49787 / 構造因子数: 6282 / 位相誤差の除外基準: 0 / Rmerge: 33.5 |
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解析
EM 3D crystal entity | ∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 42.74 Å / B: 48.96 Å / C: 99.64 Å / 空間群名: P212121 / 空間群番号: 19 | |||||||||||||||||||||||||||||||||||
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CTF補正 | タイプ: NONE | |||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 2.5 Å / 解像度の算出法: DIFFRACTION PATTERN/LAYERLINES / 対称性のタイプ: 3D CRYSTAL | |||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 9PAP Accession code: 9PAP / Source name: PDB / タイプ: experimental model | |||||||||||||||||||||||||||||||||||
精密化 | 構造決定の手法: ![]()
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溶媒の処理 | 減衰半径: 0.9 Å / VDWプローブ半径: 1.1 Å / 溶媒モデル: FLAT BULK SOLVENT MODEL | |||||||||||||||||||||||||||||||||||
拘束条件 |
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LS精密化 シェル |
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