ジャーナル: Science / 年: 2019 タイトル: Structural basis of α-scorpion toxin action on Na channels. 著者: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio ...著者: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio Ciferri / Christopher A Ahern / Frank Bosmans / David H Hackos / Alexis Rohou / Jian Payandeh / 要旨: Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na ...Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Na channels.
モデル: C-flat-2/1 / 材質: COPPER / メッシュ: 200 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY ARRAY / 前処理 - タイプ: PLASMA CLEANING 詳細: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were ...詳細: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were then coated on both sides with 5nm of Au/Pd which was plasma deposited using the Leica ACE600 (Leica).
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV
詳細: Automated picking using a soft-edged disc as a template yielded 842,024 candidate coordinates. 2D classification led to the selection of 504,252 particles for further analysis.
詳細: The startup map was low-pass filtered to 40A and resolution-limited automatic refinement in cisTEM was used (beginning at 20A)
最終 再構成
使用したクラス数: 1 / 想定した対称性 - 点群: C1 (非対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: cisTEM 詳細: No information from spatial frequencies higher than 1/5A were ever used during refinement. 使用した粒子像数: 251714