ジャーナル: Nat Commun / 年: 2020 タイトル: Structural basis for tuning activity and membrane specificity of bacterial cytolysins. 著者: Nita R Shah / Tomas B Voisin / Edward S Parsons / Courtney M Boyd / Bart W Hoogenboom / Doryen Bubeck / 要旨: Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all ...Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.
詳細: Grids were glow discharged before application of graphene oxide, then left to dry for 1 hour before use. グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3
急速凍結
装置: FEI VITROBOT MARK III / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 294 K / 詳細: Wait time of 60 s, blot time 2.5 s, blot force 3
平均露光時間: 1 sec. / 電子線照射量: 66 e/Å2 / 検出モード: INTEGRATING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 詳細: Some images were collected at a 30 degree tilt. Images were collected in movie mode at 39 frames per second.
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解析
EMソフトウェア
ID
名称
バージョン
カテゴリ
2
crYOLO
粒子像選択
3
EPU
画像取得
5
CTFFIND
4
CTF補正
8
PHENIX
モデルフィッティング
9
Coot
モデルフィッティング
11
RELION
初期オイラー角割当
12
RELION
最終オイラー角割当
13
RELION
分類
14
RELION
3次元再構成
15
PHENIX
モデル精密化
CTF補正
タイプ: NONE
3次元再構成
解像度: 4.6 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 51041 / クラス平均像の数: 2 / 対称性のタイプ: POINT
原子モデル構築
プロトコル: RIGID BODY FIT / 空間: REAL 詳細: Model of ILY-CD59 was fit and refined into the central subunit density. A combination of rigid body fitting and global minimization with secondary structure restraints was used to fit and ...詳細: Model of ILY-CD59 was fit and refined into the central subunit density. A combination of rigid body fitting and global minimization with secondary structure restraints was used to fit and refine the central subunit model. Then the central subunit model was rigid body fit as one body into the neighbouring subunits to generate a 3 subunit oligomer model.