In silico reduction of conformational heterogeneity for single-particle cryo-EM

单粒子低温电子显微镜已成为研究大分子及其复合物结构的有力工具。 冷冻EM图像分析的最大挑战之一是分子的异质性和灵活性，这严重限制了可实现的分辨率。 在建议的项目中，我们将开发一种计算方法来减少一组单粒子图像的构象方差，以提高分辨率。我们提出了一种方法，是基于一种方法来提取构象波动的自举程序，我们已经开发了较早。 然后使用关于构象方差的信息将图像广泛地分类成大量不同的类。 在下一步骤中，通过适当的平均过程将来自所有类别的个体3D密度重建组合成一个单个重建。 目的是提高分辨率，同时获得大分子构象变化的完整图像。然后将所提出的方法应用于两个不同的系统：1）与Holger Stark小组合作，我们将研究SelB-核糖体复合物，其中负责核糖体识别的SelB结构域4由于高度的异质性而目前无法解析到高分辨率。 2）与Wah Chiu小组一起，我们将致力于GroEL/ES-RuBisCO综合体。我们最近发表了这种复合物的结构，显示封装的基板，这似乎是几种构象的混合物。 我们的目标是分离这些不同的构象，并确定GroEL/ES腔内的折叠中间体的结构。

Single-particle cryo-electron microscopy has become a powerful tool to study the structure of large macromolecules and complexes thereof. One of the biggest challenges in the analysis of cryo-EM images is the heterogeneity and flexibility of the molecules, which severely limits the achievable resolution. In the proposed project we will develop a computational approach to reduce the conformational variance of a set of single-particle images with the goal of increasing the resolution. We propose an approach that is based on a method to extract conformational fluctuations from a bootstrapping procedure, which we have developed earlier. The information on conformational variance is then used to extensively classify the images into a large number of different classes. In a next step, the individual 3D density reconstructions from all classes are combined into one single reconstruction by an appropriate averaging procedure. The goal is to improve the resolution and at the same time to gain a complete picture of the conformational variance of the macromolecule. The proposed method will then be applied to two different systems: 1) in collaboration with the group of Holger Stark we will study the SelB-ribosome complex, where domain 4 of SelB, which is responsible for recognition by the ribosome, at the moment cannot be resolved to high resolution due to a high degree of heterogeneity. And 2), with the group of Wah Chiu we will work on the GroEL/ES-RuBisCO complex. We recently published a structure of this complex showing the encapsulated substrate, which seems to be a mixture of several conformations. We aim to separate these different conformations and determine the structure of a folding intermediate within the GroEL/ES cavity.