Time-resolved cryo-electron microscopy: Recent progress

Abstract

Time-resolved cryo-electron microscopy (cryo-EM) combines the known advantages of single-particle cryo-EM in visualizing molecular structure with the ability to dissect the time progress of a reaction between molecules in vitro. Here some of the recent progress of this methodology and its first biological applications are outlined.

Keywords: Cryo-EM; Microfluidics; RRF; Ribosome recycling; Short-lived states.

Fig. 1

Apparatus for time-resolved cryo-EM. (a) Close-up of the apparatus, and further close-up on the mounted chip with four inlets. The two inlets on the left conduct the two reactants into the chip; the two on the right both carry nitrogen gas under pressure, for operating the sprayer. (b) Schematic diagram of the microfluidic chip. (Reproduced with permission from Chen and Frank, 2016).

Fig. 2

The process of of ribosome recycling. (A) Flow diagram depicting the reactions following the mixing of the starting complex PostTC•RRF with EF-G in the presence of GTP. (The branch on the left represents a non-productive path in which RRF falls off and is replaced by EF-G.) (B) Graph of kinetic measurements. The fraction of interesting short-lived intermediate PostTC•RRF•EF-G reaches a peak at ~70 ms and falls off within a second, bringing it out of reach of standard blotting/plunge-freezing cryo-EM. The time point caught by the microfluidic chip is indicated with a dashed line. The black curve depicts the rise in the fraction of separate 50S and 30S subunits. (Adapted from Fu et al., 2016).

Fig. 3

Inventory of complexes found at 140 ms (complexes 1, 3–6), spray control (complex 2), and long-exposure control (complex 7). Ligands segmented out: EF-G (dark blue), RRF (red), E-site tRNA (orange), IF3 (brown). Resolution is stated in angstrom. The spray control sample was obtained by passing PostTC•RRF through one channel and buffer through the other. Long-exposure control was obtained by long-time (minutes) reaction and standard cryo-EM after passing the sample through one channel of the time-resolved apparatus, and buffer through the other. (Based on Fu et al., 2016).