Time-Resolved Cryo-Em Study Of Ribosome Subunit Association By Mixing-Spraying

The emergence of a high-throughput data processing pipeline and efficient classification algorithms has revived the idea of time-resolved cryogenic electron microscopy (cryo-EM), i.e., capturing time-dependent structures in a biological specimen using cryo-EM. The blotting method now routinely used to prepare cryo-EM specimen is sufficient only to study a reaction in the second to minute range. Capturing faster reactions, in the sub-second range, has been a practical challenge, due to the required step of depositing the specimen on the grid. To address this challenge, Lu et al. (2009) have developed a novel method to prepare time-resolved cryo-EM specimens, by using a nano-fabricated mixing-spraying chip. The mixing-spraying chip allows a two-component reaction to proceed inside the chip for tens to hundreds of milliseconds (ms), which is then stopped by fast freezing. Here we used the improved mixing-spraying method to study ribosome subunit association, and particularly to capture the conformational changes of the ribosome in this reaction. Ribosome subunit association is a pivotal step in translation initiation. Previous ensemble kinetic studies suggested that the ribosome subunit association is a multi-step process, with the ribosomal inter-subunit bridges sequentially formed. Using time-resolved cryo-EM, we were able to capture the association reaction in a pre-equilibrium state by mixing the two ribosomal subunits and reacting for 140ms. However, the fraction population of the associated ribosomes in each conformation was stable from 140ms to 75min, indicating that the ribosome undergoes conformational changes faster than the 140ms time frame upon association of the subunits. Thus, we have demonstrated that the mixing-spraying method of time-resolved cryo-EM is able to visualize the states of macromolecules in a reaction within a sub-second time frame.