摘要
冷冻电镜单颗粒三维重构方法解析生物大分子结构通常需要收集大量的照片进行分析。本工作证明当冷冻样品质量足够好时,仅使用单张冷冻电镜显微照片上的蛋白颗粒图像就可以获得近原子级分辨率的单颗粒三维重构密度图,并分别解析了apoferritin和20S蛋白酶体的三维结构。研究发现,由于蛋白分子颗粒在样品中Z轴不同位置的分布和电子显微镜的像散作用对单张照片中衬度传递函数零点缺失形成了综合补偿效应,从而在单颗粒三维重构中有效恢复了空间频率全域内的结构信息。本研究通过分析不同累计曝光剂量数据的三维重构发现,保存无损高频信息以及足够角度搜索的低频信息的最低辐照剂量其实远低于当前通用的累积曝光量。据此,本工作估算了完成正确高分辨率三维结构解析所需要的最低辐照剂量以及最小颗粒数。本工作加深了我们对冷冻电镜数据信号在图像中保存的理解,为开发更好的冷冻电镜数据采集策略、更好地保存完整高频信号提供了实验上的启示。
Single⁃particle Cryo⁃electron microscopy(Cryo⁃EM)typically requires a large number of images to reconstruct the three⁃dimensional structures of biological macromolecules.This study demonstrates that near⁃atomic resolution 3D reconstructions can be achieved using protein particles from a single Cryo⁃EM micrograph with optimal sample quality.We successfully resolved the 3D structures of apoferritin and the 20S proteasome from each single micrograph.Our findings suggest that the combined effects of Z⁃axis distribution of protein particles and astigmatism correction allow effective recovery of structural information across the whole spatial frequency domain,especially in the presence of contrast transfer function(CTF)zero⁃crossings.Furthermore,by analyzing reconstructions at varying cumulative electron doses,we identified that the minimum dose required to preserve high⁃frequency information and ensure adequate angular sampling with low⁃frequency information is significantly lower than current standards.Based on these results,we estimated the minimal radiation dose and particle count necessary for high⁃resolution 3D structure determination.This study improves our understanding of signal preservation in Cryo⁃EM imaging and provides experimental insights for optimizing data collection strategies aimed at preserving complete high⁃frequency information.
作者
杨梓
范潇
王宏伟
YANG Zi;FAN Xiao;WANG Hongwei(School of Life Sciences Tsinghua University,Beijing 100084;Beijing Frontier Research Center for Biological Structure,Beijing 100084;Tsinghua-Peking Joint Center for Life Sciences,Beijing 100084,China)
出处
《电子显微学报》
北大核心
2025年第1期91-105,共15页
Journal of Chinese Electron Microscopy Society
基金
国家自然科学基金资助项目(H.-W.W.No.31825009).
