+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6on2 | ||||||||||||||||||
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タイトル | Lon Protease from Yersinia pestis with Y2853 substrate | ||||||||||||||||||
要素 |
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キーワード | HYDROLASE / Lon / mitochondrial protease / AAA+ / ATPase | ||||||||||||||||||
機能・相同性 | 機能・相同性情報 endopeptidase La / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / cellular response to heat / sequence-specific DNA binding / serine-type endopeptidase activity / ATP hydrolysis activity / ATP binding / cytoplasm 類似検索 - 分子機能 | ||||||||||||||||||
生物種 | Yersinia pestis (ペスト菌) | ||||||||||||||||||
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 Å | ||||||||||||||||||
データ登録者 | Shin, M. / Asmita, A. / Puchades, C. / Adjei, E. / Wiseman, R.L. / Karzai, A.W. / Lander, G.C. | ||||||||||||||||||
資金援助 | 米国, 5件
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引用 | ジャーナル: Sci Adv / 年: 2020 タイトル: Structural basis for distinct operational modes and protease activation in AAA+ protease Lon. 著者: Mia Shin / Cristina Puchades / Ananya Asmita / Neha Puri / Eric Adjei / R Luke Wiseman / A Wali Karzai / Gabriel C Lander / 要旨: Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate ...Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates. | ||||||||||||||||||
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 | 6on2.cif.gz | 526.1 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6on2.ent.gz | 436.2 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6on2.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 | 6on2_validation.pdf.gz | 1.7 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 6on2_full_validation.pdf.gz | 1.8 MB | 表示 | |
XML形式データ | 6on2_validation.xml.gz | 86.1 KB | 表示 | |
CIF形式データ | 6on2_validation.cif.gz | 127.7 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/on/6on2 ftp://data.pdbj.org/pub/pdb/validation_reports/on/6on2 | 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: 57927.051 Da / 分子数: 6 / 由来タイプ: 組換発現 / 由来: (組換発現) Yersinia pestis (ペスト菌) / 遺伝子: lon, YP_0776, EGT45_05820, NCTC144_01047 / 発現宿主: Escherichia coli (大腸菌) 参照: UniProt: A0A3N4AY83, UniProt: A0A5P8YJ65*PLUS, endopeptidase La #2: タンパク質・ペプチド | | 分子量: 515.560 Da / 分子数: 1 / 由来タイプ: 組換発現 詳細: Y2853 substrate was added to Lon and modeled here as a polyalanine chain 由来: (組換発現) Yersinia pestis (ペスト菌) / 発現宿主: Escherichia coli (大腸菌) #3: 化合物 | ChemComp-ATP / #4: 化合物 | ChemComp-MG / #5: 化合物 | |
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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: Lon protease bound to Y2853 substrate / タイプ: COMPLEX 詳細: Complexes consisting of homohexameric Lon protease from Yersinia pestis bound to Y2853 substrate were isolated using size-exclusion chromatography Entity ID: #1-#2 / 由来: RECOMBINANT | ||||||||||||||||||||||||||||||
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分子量 | 単位: MEGADALTONS / 実験値: YES | ||||||||||||||||||||||||||||||
由来(天然) | 生物種: Yersinia pestis (ペスト菌) / 細胞内の位置: Cytoplasm | ||||||||||||||||||||||||||||||
由来(組換発現) | 生物種: Escherichia coli (大腸菌) / プラスミド: pET28b-lon | ||||||||||||||||||||||||||||||
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. Buffers were stored on ice and used within 15 minutes of mixing in ...詳細: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Buffers were stored on ice and used within 15 minutes of mixing in order to avoid excess ATP hydrolysis. | ||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.95 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: Grids were plasma treated for 30 seconds using a 15 mA current operating under atmospheric gases using a glow discharger (Electron Microscopy Sciences). 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: Quantifoil, 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 sec. / 電子線照射量: 52 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: 2 / 実像数: 4071 詳細: Images were collected in counting mode at 4 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: 44 / 利用したフレーム数/画像: 0-43 |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.11.1_2580: / 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 correction in RELION / タイプ: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 1176206 / 詳細: template-based cross correlation with FindEM | ||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 118143 / アルゴリズム: BACK PROJECTION 詳細: Focused classification of final reconstruction was performed on E and F "step" subunits, resulting in a reconstruction with an overall resolution of 3.5 A by FSC 0.143. The two maps were ...詳細: Focused classification of final reconstruction was performed on E and F "step" subunits, resulting in a reconstruction with an overall resolution of 3.5 A by FSC 0.143. The two maps were stitched together using vop max in UCSF Chimera. All three maps (two original and final composite) are deposited in this entry. クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 52 / プロトコル: AB INITIO MODEL / 空間: REAL / Target criteria: Correlation coefficient 詳細: Initial homology model was built using SWISS-MODEL and initial rigid body docking was done using UCSF Chimera | ||||||||||||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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