National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
米国
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)
米国
引用
ジャーナル: Sci Adv / 年: 2021 タイトル: Structural insights into integrin αβ opening by fibronectin ligand. 著者: Stephanie Schumacher / Dirk Dedden / Roberto Vazquez Nunez / Kyoko Matoba / Junichi Takagi / Christian Biertümpfel / Naoko Mizuno / 要旨: Integrin αβ is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and αβ undergo conformational changes. While this is key for cell-tissue connections, ...Integrin αβ is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and αβ undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human αβ with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The αβ-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting αβ adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of αβ for fibronectin is increased with manganese ions (Mn) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and αβ opening is induced by ligand-binding.
凍結剤: ETHANE-PROPANE / 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
Integrin a5b1 was assembled into MSPE3D1 nanodiscs that contained bovine brain lipids (Folch fraction I) at a ratio of 1:29:3460, respectively. The assembly mix was incubated with SM-2 BioBeads to remove DDM detergent from solubilized lipids and then purified by size-exclusion chromatography using a Superose 6 3.2/300 column.
initial image processing automatically by FOCUS pipeline: gain normalization, motion correction and dose-weighting in MotionCor2 CTF estimation by GCTF
粒子像選択
選択した数: 496356 詳細: 1424 particles were manually picked and used to obtain 2D classes with distinct features as templates for Gautomatch
CTF補正
ソフトウェア - 名称: Gctf
初期モデル
モデルのタイプ: OTHER 詳細: 30 A-low pass filtered model from previous data analysis: 502 particles were manually picked from 2600 micrographs and used for 2D classification to generate a template for gautomatch, which ...詳細: 30 A-low pass filtered model from previous data analysis: 502 particles were manually picked from 2600 micrographs and used for 2D classification to generate a template for gautomatch, which picked 103534 particles. After 2D classification an initial model was obtained in Cryosparc. Further 3D classification and refinement in Relion used 23332 particles yielded a 3D map that was used as a starting model for the high-resolution data set.