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Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

Significance The protein-synthesizing machinery of the cell, the ribosome, is made up of two subunits, which in bacteria are held together by 12 molecular bridges. Understanding the mechanism and timeline of bridge formation is crucial to understanding the mechanism of bacterial translation initiation. To study the timeline of bridge formation, we developed microfluidic devices that mix two interacting components, spray the mixture onto an electron microscopy grid, and flash freeze the grid for a minimum reaction time of 9.4 ms. This study shows for the first time to the authors' knowledge that eight of the 12 bridges form within 9.4 ms, whereas the remaining four bridges take longer than 43 ms to form. Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA–RNA, RNA–protein, and protein–protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.