+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6zxj | ||||||
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タイトル | Fully-loaded anthrax lethal toxin in its heptameric pre-pore state, in which the third lethal factor is masked out (PA7LF3-masked) | ||||||
要素 |
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キーワード | TOXIN / anthrax lethal toxin / fully-loaded pre-pore state / membrane translocase / cytotoxic substrate | ||||||
機能・相同性 | 機能・相同性情報 anthrax lethal factor endopeptidase / positive regulation of apoptotic process in another organism / host cell cytosol / negative regulation of MAPK cascade / Uptake and function of anthrax toxins / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity ...anthrax lethal factor endopeptidase / positive regulation of apoptotic process in another organism / host cell cytosol / negative regulation of MAPK cascade / Uptake and function of anthrax toxins / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / identical protein binding / membrane / metal ion binding 類似検索 - 分子機能 | ||||||
生物種 | Bacillus anthracis (炭疽菌) | ||||||
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: 3.5 Å | ||||||
データ登録者 | Quentin, D. / Antoni, C. / Gatsogiannis, C. / Raunser, S. | ||||||
資金援助 | ドイツ, 1件
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引用 | ジャーナル: PLoS Pathog / 年: 2020 タイトル: Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state. 著者: Claudia Antoni / Dennis Quentin / Alexander E Lang / Klaus Aktories / Christos Gatsogiannis / Stefan Raunser / 要旨: Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA) ...Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first. | ||||||
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 | 6zxj.cif.gz | 870.3 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6zxj.ent.gz | 699.4 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6zxj.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 | 6zxj_validation.pdf.gz | 882.1 KB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 6zxj_full_validation.pdf.gz | 989.2 KB | 表示 | |
XML形式データ | 6zxj_validation.xml.gz | 138.6 KB | 表示 | |
CIF形式データ | 6zxj_validation.cif.gz | 210.4 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/zx/6zxj ftp://data.pdbj.org/pub/pdb/validation_reports/zx/6zxj | 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: 85679.930 Da / 分子数: 7 / 由来タイプ: 組換発現 / 由来: (組換発現) Bacillus anthracis (炭疽菌) / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: Q68GS1, UniProt: P13423*PLUS #2: タンパク質 | 分子量: 93904.211 Da / 分子数: 2 / 由来タイプ: 組換発現 / 由来: (組換発現) Bacillus anthracis (炭疽菌) / 遺伝子: lef, pXO1-107, BXA0172, GBAA_pXO1_0172 / 発現宿主: Bacillus anthracis (炭疽菌) 参照: UniProt: P15917, anthrax lethal factor endopeptidase |
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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 |
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分子量 |
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由来(天然) |
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由来(組換発現) |
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緩衝液 | pH: 8.5 | ||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.06 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||
急速凍結 | 装置: GATAN CRYOPLUNGE 3 / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 286 K 詳細: 4 uL sample was applied to grid (with 2 nm additional carbon layer) and incubated for 45 s prior blotting. |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 130000 X / Calibrated defocus min: 1200 nm / 最大 デフォーカス(補正後): 2600 nm / Cs: 2.7 mm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 平均露光時間: 15 sec. / 電子線照射量: 74.4 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) 実像数: 5238 |
電子光学装置 | エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 20 eV |
画像スキャン | 動画フレーム数/画像: 40 |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.18.2_3874: / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 382000 | ||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||||||||||||||||||
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: 210000 / アルゴリズム: BACK PROJECTION / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT 詳細: The protective antigen (PA) monomer from 3HVD was placed seven times into the density corresponding to the heptameric PA ring using the rigid-body fit in Chimera. Two copies of the monomeric ...詳細: The protective antigen (PA) monomer from 3HVD was placed seven times into the density corresponding to the heptameric PA ring using the rigid-body fit in Chimera. Two copies of the monomeric lethal factor (LF) from 1J7N were fitted similarly into the corresponding LF density located atop of the PA7 ring. The resulting model was then flexibly fitted using iMODFIT. The model was further refined in iterative rounds of phenix and coot. Unresolved regions were deleted and side chain information was removed for less well-defined regions. | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | 3D fitting-ID: 1 / PDB chain-ID: A / Source name: PDB / タイプ: experimental model
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拘束条件 |
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