Yapici, I.; Dao, E. H.; Yokoi, S.; Destan, E.; Ayan, E.; Shafiei Kamel, A.; Ertem, F. B.; Kulakman, C.; Yilmaz, M.; Tosun, B.; Ciftci, H.; Kepceoglu, A.; Johnson, J. A.; Guven, O.; Ergul, A.; Hayes, B.; Rao, Y.; Kupitz, C. J.; Poitevin, F.; Liang, M.; Hunter, M. S.; Milon, P.; Mitsutake, A.; Sierra, R. G.; Wakatsuki, S.; DeMirci, H.

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.