a, b, 3D cryo-EM-derived Coulomb potential maps30 of the 30S IC (a) and the 30S subunit + fMet-tRNAfMet complex (b) obtained from a control experiment in which the 30S IC in Tris-polymix buffer and a solution of Tris-polymix buffer lacking 50S subunits were injected into the microfluidic chip designed to give the longest reaction time (~600 ms), mixed, allowed to react, and sprayed onto an electron microscopy grid that was rapidly plunged into liquid ethane. The sizes of the resulting populations of the 30S IC and the 30S subunit + fMet-tRNAfMet complex were 75% and 25%, respectively, which demonstrates that most of the 30S ICs remain intact during the mixing-spraying process. c, Plot of the concentrations of the 50S subunit, 70S IC and 70S EC as a function of time generated by using the initial 50S subunit and 30S IC concentrations analogous to those used in our mixing-spraying microfluidic chip (that is, 0.6 µM and 1.2 µM, respectively) and modelling the kinetics of subunit joining using the kinetic scheme and set of rate constants reported previously for a subunit-joining reaction performed in the presence of IF1 and IF2, but in the absence of the IF319. A detailed description of the kinetic modelling can be found in the Methods. The plot predicts that the 70S IC population should peak within 50–250 ms after mixing of the 50S subunit and 30S IC, and that these 70S ICs should mature to a notable population of 70S ECs within the next several hundreds of milliseconds. Therefore, to ensure that we would capture formation of the 70S IC and its maturation to the 70S EC, we selected microfluidic chips designed to provide reaction times of approximately 20 ms, 80 ms, 200 ms and 600 ms. The free 50S subunit, 70S IC and 70S EC populations observed in our time-resolved cryo-EM experiments are shown as blue diamonds, light grey circles and dark grey triangles, respectively.