Cryo-EM 3D Reconstruction of Flexible Particles

描述(申请人提供)：单粒子重建(SPR)电子显微镜是一种强大的技术，可以获得近自然状态下的大分子络合物的三维结构。SPR的一个主要问题是，当大分子粒子冻结在玻璃冰中时，弹性会产生多种构象。传统的SPR假设大分子粒子是刚性的，只有当所有粒子具有相同的结构时才能给出良好的重建。我们建议开发新的重建方法，完全消除刚性假设。这些方法可以重建连续柔性结构。提出了两类方法：(A)探索性分析方法，其检测结构构象变化的存在并客观地确定最主要的柔韧性模式；(B)探索性后柔性粒子重建方法，其详细重建已识别的柔韧性模式。这些方法是基于概率论的，实际数据的初步结果表明，即使在数据信噪比低至-25db的情况下，它们也能提供明显更高的信噪比(SNR)重建。此外，我们的方法提供了对粒子灵活性的简单解释。我们将开发这些算法并编写软件模块，以便整合到SPARX Cryo-EM处理环境中。作为第一个应用，我们将使用该软件处理人类DICER RNA加工酶的负染和冷冻EM图像。作为阳性对照和阴性对照，我们还将重建RNA聚合酶II和GroEL的结构。

DESCRIPTION (provided by applicant): Single-particle reconstruction (SPR) electron microscopy is a powerful technique for obtaining the 3D structure of macromolecular complexes in near-native states. A major problem in SPR is that flexibility gives rise to multiple conformations when the macromolecular particles are frozen in vitreous ice. Traditional SPR assumes that macromolecular particles are rigid, and gives good reconstructions only when all particles have the identical structure. We propose to develop new reconstruction methods which eliminate the rigidity assumption altogether. These methods can reconstruct a continuously flexible structure. Two classes of methods are proposed: (a) methods for exploratory analysis which detect the presence of structural conformational change and objectively determine the most dominant modes of flexibility, and (b) post- exploratory flexible particle reconstruction methods which reconstruct in detail the identified modes of flexibility. These methods are based on probability theory and preliminary results with actual data show that they offer significantly higher signal-to-noise ratio (SNR) reconstructions even when the data SNR is as low as -25db. Further, our methods provide a simple interpretation of particle flexibility. We will develop these algorithms and write software modules for incorporation in the SPARX cryo-EM processing environment. As a first application, we will use the software to process negative-staining and cryo-EM images of the human Dicer RNA-processing enzyme. As positive and negative controls, we will also reconstruct the structure of RNA Polymerase II and GroEL.