Structural biology in cellulo: Minding the gap between conceptualization and realization

Recent technological advances have deepened our perception of cellular structure. However, most structural data doesn't originate from intact cells, limiting our understanding of cellular processes. Here, we discuss current and future developments that will bring us towards a structural picture of the cell. Electron cryotomography is the standard bearer, with its ability to provide in cellulo snapshots. Single-particle electron microscopy (of purified biomolecules and of complex mixtures) and covalent crosslinking combined with mass spectrometry also have significant roles to play, as do artificial intelligence algorithms in their many guises. To integrate these multiple approaches, data curation and standardisation will be critical – as is the need to expand efforts beyond our current protein-centric view to the other (macro)molecules that sustain life. •AI tools like AlphaFold revolutionize protein structure prediction and analysis.•Atomic resolution with cryoET in cells is feasible – but remains a challenge.•Single-particle cryoEM gives atomic resolution but lacks spatial information.•Spatial information for protein interactions is recovered by crosslinking MS.•Analyzing diverse macromolecules in cells is a significant challenge.