Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells

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 3D 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 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.