Recent advances in cryo electron microscopy (cryo-EM) and image processing provide new opportunities to analyse drug targets at high resolution. However, structural heterogeneity limits resolution in many practical cases, hence restricting the level at which structural details can be analysed and drug design be performed. As structural disorder is not spread throughout the entire structure of a given macromolecular complex but instead is found in certain regions that move with respect to others and covering molecular scales from domain conformational changes up to the level of side chain conformations in ligand binding pockets, it is possible to focus the attention on those regions and the associated relative movements. Here we show how the usage of focused classifications and refinements provide insights into global conformational arrangements, exemplified on the human ribosome and on the cannabinoid G protein coupled receptor (GPCR), and how they can improve the local map resolution from an essentially disordered region to the 3–4 Å and finally to the 2 Å resolution range. A systematic analysis with variable spherical masks during focused refinement is presented showing that the choice of an optimal mask size helps refining to high resolution. This study covers several practical approaches on 4 examples illustrating how important mask size & shape and including neighbouring structural elements are for a focused analysis of a macromolecular complex. Such methods will be crucial for cryo-EM structure-based drug design of various medical targets and are applicable to single particle cryo-EM and electron tomography data.

冷冻电子显微镜 (cryo-EM) 和图像处理的最新进展为高分辨率分析药物靶标提供了新的机会。然而，结构异质性在许多实际情况下限制了分辨率，从而限制了结构细节分析和药物设计的水平。由于结构紊乱并未遍布给定大分子复合物的整个结构，而是存在于相对于其他区域移动的某些区域中，并且覆盖了从结构域构象变化到配体结合口袋中侧链构象水平的分子尺度，因此可以将注意力集中在这些区域和相关的相对运动上。在这里，我们展示了如何使用集中分类和细化来深入了解全局构象排列，以人类核糖体和大麻素 G 蛋白偶联受体 (GPCR) 为例，以及它们如何将局部图谱分辨率从本质上无序的区域提高到3-4 Å 分辨率范围，最后达到 2 Å 分辨率范围。提出了在聚焦细化过程中使用可变球形掩模的系统分析，表明最佳掩模尺寸的选择有助于细化到高分辨率。这项研究涵盖了 4 个示例的几种实用方法，说明了掩模尺寸和形状以及包括相邻结构元素对于大分子复合物的集中分析的重要性。这些方法对于各种医学靶点的基于冷冻电镜结构的药物设计至关重要，并且适用于单颗粒冷冻电镜和电子断层扫描数据。