Assembly of anthrax toxin pore: Lethal-factor complexes into lipid nanodiscs
Assembly of anthrax toxin pore: Lethal-factor complexes into lipid nanodiscs
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DOI：
10.1002/pro.2231
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发表时间：
2013-04-01
期刊：
影响因子：
8
通讯作者：
Fisher, M. T.
中科院分区：
文献类型：
--
作者：
Akkaladevi, N.;Hinton-Chollet, L.;Fisher, M. T.
我们已经设计了一个程序，将炭疽保护性抗原（PA）孔与炭疽致死因子（LFN）的N-末端结构域复合到脂质纳米盘中，并通过负染电子显微镜分析所得复合物。在不依赖初级和次级洗涤剂筛选的情况下进行插入纳米盘。制备物相对纯净，并且插入EM网格上的纳米盘中的PA孔的百分比很高（约43%）。负染色的单个颗粒的三维分析揭示了LFN-PA纳米盘复合物反映了先前的未配体的PA孔纳米盘结构，但具有与PA帽区域上的多个结合的LFN分子一致的额外的蛋白质密度。组装过程将有助于收集更高分辨率的cryo-EM LFN-PA纳米盘结构和使用先进的自动化颗粒选择方法。
We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (approximate to 43%). Three-dimensional analysis of negatively stained single particles revealed the LFN-PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN-PA nanodisc structures and use of advanced automated particle selection methods.