Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and inside cells. In addition to aligning individual particles, accurate registration of sample motion and three-dimensional deformation during exposures are crucial for achieving high-resolution reconstructions. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and couples a comprehensive spatial deformation model to in silico correction of electron-optical aberrations. M provides a unified optimization framework for both frame-series and tomographic tilt-series data. We show that tilt-series data can provide the same resolution as frame-series data on a purified protein specimen, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M resolves to residue level a 70S ribosome bound to an antibiotic inside intact bacterial cells. Our work provides a computational tool that facilitates structural biology in cells.

冷冻电子显微镜 (cryo-EM) 可在体外和细胞内测定大分子结构。除了对齐单个粒子外，准确记录样本运动和曝光期间的三维变形对于实现高分辨率重建至关重要。在这里，我们描述了 M，这是一种软件工具，它为低温 EM 数据建立基于参考的多粒子细化框架，并将综合空间变形模型与电子光学像差的计算机校正相结合。M 为帧序列和层析倾斜序列数据提供统一的优化框架。我们表明，倾斜系列数据可以提供与纯化蛋白质标本上的帧系列数据相同的分辨率，表明对齐步骤不再限制从断层扫描数据中获得的分辨率。结合 Warp 和 RELION，M 将与完整细菌细胞内的抗生素结合的 70S 核糖体分解为残留水平。我们的工作提供了一种促进细胞结构生物学的计算工具。