Initial bridges between the two ribosomal subunits are formed within 9.4 milliseconds: A time-resolved cryo-EM study

Tanvir R. Shaikh^1,2, Aymen Yassin^2,5, Zonghuan Lu³, David Barnard², Xing Meng², Toh-Ming Lu³, Terence Wagenknecht^2,4, and Rajendra K. Agrawal^2,4

¹CEITEC, Masaryk University, Brno, Czech Republic
²Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201

³Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180

⁴Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY
⁵Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

The bacterial 70S ribosome are held together by 13 dynamic bridges between its two subunits (30S and 50S) involving RNA-RNA, RNA-protein, and protein-protein interactions. We have developed and implemented a class of microfluidic devices that mixes two components to completion within 0.4 milliseconds (ms) and sprays the mixture in the form of microdroplets onto a cryo-electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryo-fixation. We have used such devices to study association between the two E. coli ribosomal subunits, by collecting cryo-EM data corresponding to reaction times of 9.4 and 43 ms. According to our image analysis, about 25% of ribosomal subunits are already engaged in the formation of 70S ribosome particles within 9.4 ms, and at 43 ms, 49% of the ribosomal subunits have undergone formation of 70S ribosomes. Molecular analysis of the corresponding three-dimensional reconstructions suggests that the inter-subunit bridges B2a, B2b, B3, and B7a form within 9.4 ms and bridges such as bridges B2c, B4, B5, and B6 take longer than 43 ms to form. This approach can be used to characterize the sequence of dynamic functional events on a complex macromolecular assembly, such as the ribosome.