Advancing bacterial cell biology with in-cell cryo-electron microscopy

Over the past decades, bacterial cell biology has revealed that bacteria possess complex subcellular structures and regulatory mechanisms once thought to be unique to eukaryotes, establishing them as powerful model systems for studying fundamental cellular principles. However, their small size makes bacterial subcellular structures difficult to image with optical microscopy. In-cell cryo-electron microscopy (cryo-EM) is a cutting-edge technique that enables high-resolution visualization of macromolecular complexes and the networks they form in their native context. In this review, we highlight the unique advantages of both cryo-electron tomography (cryo-ET) and in-cell single-particle analysis (in-cell SPA) in bacterial cell biology and summarize key advances from the past two years, including structural studies of macromolecular complexes and investigations of dynamic cellular processes, underscoring their growing importance and potential as a scaffold for multidisciplinary approaches.