Program System for Protein Electron Crystallography

二维晶体电子衍射法测定膜结合蛋白的三维结构作为X射线衍射的补充方法在结构生物学中具有重要意义。本工作的目的是开发一个电子晶体学的建模和精化工具。通过研究，我们确定了各种蛋白质的三维结构，并建立了建模和细化技术。技术发展的结果可以在细菌视紫红质和水通道蛋白的原子分辨率模型中看到，这些模型提供了关于其功能如何在3D结构上发生的机制的重要信息。从分析中可以清楚地看出，电子衍射中最基本的特征是有关蛋白质带电状态的信息。在X射线散射中，电子被电子势散射而不是被电子密度散射。因此，价电子强烈地影响电子的散射行为，换句话说，电子衍射依赖于组成原子的带电状态。我们发展了一种正确处理带电状态的方法，方法如下。我们对构成蛋白质分子的小分子模型化合物在不同带电状态和溶剂化状态下的电子势进行了从头计算，并通过对势函数的傅里叶变换计算了每个带电状态和溶剂化状态下的原子散射因子。在结构分析的应用中，由于电荷状态在晶体中具有一定的分布，因此在细化过程中将计算各种电荷状态的原子散射因子的最佳线性组合。

3D Structure determination of membrane bound proteins by electron diffraction from 2D crystal has a crucial importance in structural biology as a complementary method to X-ray diffraction. The purpose of this work is to develop a modeling and refinement tool for the electron crystallography. Through the research period, we have determined 3D structures of various proteins, and established the modeling and refinement techniques. The results of the technical development can be seen in the atomic resolution models of bacteriorhodopsin and aquaporin, which provide important pieces of information about the mechanism how their functions occur on the 3D structures. It has become clear from the analyses that the most fundamental characteristics in electron diffraction is in the information about the charged state of a protein. Electron is scattered by the electronic potential instead of electron density in X-ray scattering. Hence, the valence electron strongly affects the scattering behavior of electron. In other words, the electron diffraction depends on the charged state of the constituent atoms. We developed a method of treating charged state correctly by the following way. We carred out ab initio quantum chemical calculations of the electronic potentials of small model compounds, which constitute a protein molecule, in various charged states and solvation states, and calculated the atomic scattering factor for each charged and solvation state by the Fourier transformation of the potential function. In the application to the structural analysis, since the charge state has a certain distribution in a crystal, an optimal linear combination of the atomic scattering factors for various charge states will be calculated in the course of the refinement process.