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

Abstract

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.

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

“…This series of observation in Chen et al [30] is in good agreement with the results of a fast chemical probing assay [41] and a number of single-molecular fluorescence resonance energy transfer studies [42][43][44], but disagrees with another chemical probing assay [45] and the previous timeresolved cryo-EM study [29]. In comparing the two timeresolved cryo-EM studies, the major reason why the two studies reached different conclusions is the difference in the resolutions of maps (9-12 Å in Chen et al [30] and 23-33 Å in Shaikh et al [29]), which stemmed from the difference in data yield.…”

“…While the time for mixing itself is fixed and in the range of 0.5 ms for all chips, the mean reaction time is defined by the length of a meandering reaction chamber, which in the present design can be varied in the whole range from 4 to 600 ms. The time of flight for the spray and the plunging time for the grid add another 5 to 40 ms, depending on the geometry [29,30]. The mixingspraying method has been proven to work for fast reactions involving two big molecules-the ribosomal subunits [29,30] (see Case study and future improvements of the mixing-spraying method section for more details).…”

“…Both Shaikh et al [29] and Chen et al [30] studied the ribosome subunit association reaction using the mixing-spraying time-resolved cryo-EM method, as a proof-of-principle of the method and a first stride towards visual-kinetic studies of fast biological processes. The association of the small and large ribosomal subunits is an essential step in the initiation of translation, a step that also involves mRNA, initiator tRNA and a number of initiation factors in vivo (see, for example, [37][38][39][40]).…”

“…Shaikh et al analyzed cryo-EM datasets corresponding to 9 and 43 ms reaction times to identify the intermediate structures in the subunit association reaction, and proposed that eight intersubunit bridges (i.e. the contacts between the two subunits) form within 9 ms while the other four bridges take longer than 43 ms to form [29]. Chen et al [30] used 60 and 140 ms reaction times to capture the pre-equilibrium states of the subunit association reaction, as indicated by the increasing, but lower-than-equilibrium-level, population of associated ribosomes (Fig.…”

“…In comparing the two timeresolved cryo-EM studies, the major reason why the two studies reached different conclusions is the difference in the resolutions of maps (9-12 Å in Chen et al [30] and 23-33 Å in Shaikh et al [29]), which stemmed from the difference in data yield. The major reason for the low data yield in the previous applications (about 1000-2000 particles per grid) was that only the area in the thin edge of the droplets was suitable for cryo-EM data collection [25,29].…”