Visualizing chemical bonding in biological macromolecules by microcrystal electron diffraction

摘要 能够直接可视化生物分子中的化学键和电荷的方法和技术将增加 这是结构生物学领域令人兴奋的工具。与X射线不同，电子衍射对键非常敏感- 电荷分布，特别是在较低的分辨率和较小的散射角，因此电子衍射具有 用在无机样品上研究和成像化学键中的电荷分布，直接映射 既有共价键又有离子键。这种敏感度很容易从列表中的巨大差异中看出 原子和离子的电子散射因子。该项目的总体目标是开发和扩展 生物分子结构的电荷云模拟方法及其与微晶体的耦合 直接测定化合物化学键的电子衍射(MicroED)数据采集和处理 高分辨率生物分子结构。电荷云法最初是用来确定 有机小分子酞菁的成键和使用的透射电子衍射图 薄薄的晶体。结果表明，用这种方法对电子衍射数据进行适当的处理，可以得到电子的电荷密度。 在有机分子结构的二维投影中可以看到原子之间的键。要扩展这一点 方法要获得生物分子的三维结构，就需要高质量的三维电子衍射数据。 因此，我们将利用MicroED方法对几种型号的电子衍射数据进行采集 微晶样品。自发展以来，微电子放电已被证明能够产生高质量的电子 产生生物分子高分辨率3D结构的衍射数据。一种组合的方法 MicroED数据收集和处理以及低阶Bragg反射的电荷云精化将 提供了一种直接、高效、可广泛使用的技术，用于直接显示生物中的成键 样本。

SUMMARY Methods and techniques capable of directly visualizing chemical bonding and charge in biomolecules would add an exciting tool to the field of structural biology. Unlike X-rays, electron diffraction is very sensitive to the bond- charge distribution, especially at lower resolution and small scattering angles, so that electron diffraction has been used on inorganic samples to study and image the charge distribution in chemical bonds, directly mapping out both covalent and ionic bonds. This sensitivity is readily apparent from the large difference between tabulated electron scattering factors for atoms and those for ions. The overall aim of this project is to develop and extend charge-cloud modeling methods to biomolecular structures and couple these procedures with microcrystal electron diffraction (MicroED) data collection and processing for the direct determination of chemical bonding in high-resolution biomolecular structures. The charge-cloud method was initially developed to determine the bonding in an organic small molecule phthalocyanine and used transmission electron diffraction patterns from thin crystals. It was shown that by properly treating electron diffraction data with this method, charge density in the bonds between atoms could be seen in a 2D projection of the organic molecular structure. To extend this method to 3D structures of biological molecules, high-quality 3D electron diffraction data will be required. Therefore, we will make use of the MicroED method for electron diffraction data collection on several model microcrystalline samples. Since its development, MicroED has been shown to produce high-quality electron diffraction data that yields high-resolution 3D structures of biological molecules. The combined method of MicroED data collection and processing along with charge-cloud refinement of low order Bragg reflections will yield a straightforward, efficient, widely accessible technique for the direct visualization of bonding in biological samples.