In-cell nuclear magnetic resonance (NMR) spectroscopy offers the possibility to study proteins and other biomolecules at atomic resolution directly in cells. As such, it provides compelling means to complement existing tools in cellular structural biology. Given the dominance of electron microscopy (EM)-based methods in current structure determination routines, I share my personal view about the role of biomolecular NMR spectroscopy in the aftermath of the revolution in resolution. Specifically, I focus on spin-off applications that in-cell NMR has helped to develop and how they may provide broader and more generally applicable routes for future NMR investigations. I discuss the use of 'static' and time-resolved solution NMR spectroscopy to detect post-translational protein modifications (PTMs) and to investigate structural consequences that occur in their response. I argue that available examples vindicate the need for collective and systematic efforts to determine post-translationally modified protein structures in the future. Furthermore, I explain my reasoning behind a Quinary Structure Assessment (QSA) initiative to interrogate cellular effects on protein dynamics and transient interactions present in physiological environments.
Keywords: in-cell NMR; intrinsically disordered proteins; post-translational modifications; structure function; time-resolved NMR.