Conquer by cryo-EM without physically dividing.
Conquer by cryo-EM without physically dividing.
复制标题
DOI：
10.1042/bst20210360
复制
发表时间：
2021-11-01
影响因子：
3.9
通讯作者：
中科院分区：
文献类型：
--
作者：
这篇简短的综述介绍了在使用单粒子低温EM获得蛋白质和其他颗粒尺寸小于100 kDa的大分子的高分辨率结构方面所取得的实质性进展的最新进展。我们指出，在颗粒大小和可达到的分辨率方面确定所能完成的极限，对于试图使用有重点的改进方法来提高较大结构的小的柔性部分的分辨率时，可以作为预期的指南。这些方法包括在计算上忽略大分子上除特定的目标感兴趣区域之外的所有区域，被称为“掩蔽和精炼”，因此它是在计算上相当于在X射线结晶学中非常成功地使用的“分而治之”方法。然而，屏蔽精细化的好处是，人们能够在其本地建筑环境中确定结构，而无需将它们与它们发挥功能所需的生物联系物理上分开。这篇简短的综述还比较了目前的实验成果与各种理论估计的最小颗粒尺寸，这些最小颗粒尺寸可以成功地重建到高分辨率。鉴于两者之间显然仍有很大差距，我们简要回顾一下似乎有可能在设备和方法方面进一步改进的领域。
This mini-review provides an update regarding the substantial progress that has been made in using single-particle cryo-EM to obtain high-resolution structures for proteins and other macromolecules whose particle sizes are smaller than 100 kDa. We point out that establishing the limits of what can be accomplished, both in terms of particle size and attainable resolution, serves as a guide for what might be expected when attempting to improve the resolution of small flexible portions of a larger structure using focused refinement approaches. These approaches, which involve computationally ignoring all but a specific, targeted region of interest on the macromolecules, is known as ‘masking and refining,’ and it thus is the computational equivalent of the ‘divide and conquer’ approach that has been used so successfully in X-ray crystallography. The benefit of masked refinement, however, is that one is able to determine structures in their native architectural context, without physically separating them from the biological connections that they require for their function. This mini-review also compares where experimental achievements currently stand relative to various theoretical estimates for the smallest particle size that can be successfully reconstructed to high resolution. Since it is clear that a substantial gap still remains between the two, we briefly recap the areas in which further improvement seems possible, both in equipment and in methods.