Cellular structural biology probing prokaryotic and eukaryotic membrane protein complexes in-situ at atomic resolution

Cellular structural biol. aims at studying the structure and dynamics of biomols. at at. resoln. in their physiol. settings. Over the past few decades, solid-state NMR (ssNMR) has proven to be a powerful technique that can provide at.-resoln. information on membrane proteins in different lipid environments. Recently, such studies have been extended to include small membrane proteins in native milieu. However, larger complexes pose further challenges in terms of sample prepn., data anal. and sensitivity. Here, we show that sensitivity-enhanced ssNMR through Dynamic Nuclear Polarization (DNP) can be used to investigate large prokaryotic and eukaryotic membrane protein complexes in-situ at at. resoln. First, we studied the bacterial type IV secretion system core complex (T4SScc) in its native settings. T4SScc consists of three proteins, namely, VirB7, VirB9 and VirB10 that form a one megadalton complex spanning the bacterial cell envelope. By using specific-labeling with DNP-supported ssNMR, we were able to provide insight about the structure and dynamics of this complex at at. resoln. in-situ. Furthermore, we illustrate the power of this method by combining it with other biophys. and biochem. techniques to scrutinize even large eukaryotic membrane protein complexes in cellular environment. Using this approach, we were able to unravel the ligand-induced activation mechanism of the full length epidermal growth factor receptor (EGFR) in cellular milieu at at. level. Finally, we will discuss new developments in the ultrafast electron microscopy (UEM) that render it feasible to decipher the dynamics of biomols. at at. temporal resoln. (femtosecond scale). Amalgamating UEM with ssNMR/DNP in the future would allow tackling complex biol. processes at at. spatio-temporal resoln. inside the cell