考古物MCM和dsDNA复合体的开放架构,使用单分散的链状毒素亲和力CryoEMEM解决
Jianbing Ma1,2,3, Gangshun Yi1,4,5, Mingda Ye6
1Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
Nature communications
|November 28, 2024
概括
一种新的亲和网方法改善了冷电子显微镜 (cryoEM) 样本的准备,以应对具有挑战性的生物分子. 这项技术成功确定了Thermococcus kodakarensis小染色体维护复合体3 (MCM3) 的结构.
科学领域:
- 结构生物学 结构生物学
- 生物化学 生物化学
- 显微镜的使用方法
背景情况:
- 电子显微镜 (cryoEM) 对于高分辨率的生物分子结构确定至关重要.
- 样品的准备,特别是低丰度或难以获得的蛋白质,仍然是冷EM的一个重大挑战.
- 偏好的方向和空气-水接口暴露可能会阻碍数据质量.
研究的目的:
- 开发一种改进的冷EM样本制备方法,用于挑战生物分子.
- 为了增强颗粒的吸收并最大限度地减少空气-水接口效应.
- 确定Thermococcus kodakarensis小染色体维护复合体3 (MCM3) 和其复合体的结构.
主要方法:
- 在脂质单层上使用斯特雷普塔维丁开发了一种亲和网格方法.
- 使用生物化来丰富MCM3和MCM3-ATP-dsDNA复合体.
- 使用冷电子显微镜来确定结构.
主要成果:
- 通过亲和网法成功丰富并确定了MCM3的结构.
- 解决了一个稳定的MCM3-ATP-dsDNA复合物的结构.
- 对MCM3的apo和dsDNA结合状态观察到新的左侧开放螺旋形状.
- 证明了该方法与HIV-1囊蛋白和RNA聚合酶II延长复合物的实用性,缓解了偏好导向问题.
结论:
- 开发的亲和网方法显著提高了对具有挑战性的目标的冷EM样本准备.
- 新的MCM3结构为其结构状态和DNA结合/融化潜力提供了新的见解.
- 这种方法广泛适用于改善各种蛋白质复合体的冷EM研究.
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