许多大分子作为其结构和功能的特征是固有的柔性。在单粒子CryoEM处理过程中,柔性蛋白质区域可能不利于高分辨率重建,因为来自数千个粒子的信号被平均在一起。这种“模糊”效应可能难以克服,并且在平均高度对称的复合物时可能会更加明显。随着技术的进步,减轻CryoEM加工过程中灵活性的方法变得越来越重要,并应用于更动态的蛋白质和复合物。这里,我们详细介绍了使用子粒子平均和信号减法技术在设计的称为DARP14的四面体对称蛋白质支架上精确靶向和解析柔性DARPin蛋白质附件。粒子首先排列为完整的复合物,然后通过组成子单元的对齐和集中细化来降低对称性。我们获得的最终重建比完全对称重建有了很大的改进,具有可观察的二级结构和侧链位置。此外,我们还能够将脚手架的核心区域重建为2.7µ。这里概述的数据处理协议适用于其他动态和对称蛋白质复合物,我们改进的地图可以允许DARP14的新结构引导变体设计。
Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle
CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This \"blurring\" effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during
CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14.