Mix-it-up: Accessible time-resolved cryo-EM on the millisecond timescale.

Biological reactions often involve macromolecules that undergo substrate-induced conformational changes in under a second, yet capturing these transient states remains challenging. While high-resolution structural techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) have advanced our mechanistic understanding of protein-substrate interactions, traditional sample preparation methods are too slow to capture rapid biochemical events. Time-resolved cryo-EM has emerged as a promising approach to visualize structural dynamics on microsecond-to-millisecond timescales, but its widespread adoption has been limited by costly equipment and challenges in achieving rapid mixing, application, and vitrification of samples in a reproducible manner. To address these limitations, we developed "Mix-it-up" (MIU), a modified spray device designed for rapid on-grid mixing and vitrification of cryo-EM samples. By manually applying one sample onto the EM grid, blotting, and subsequently spraying the second sample, we achieve on-grid mixing with a vitrification delay of as low as ~120 milliseconds. We demonstrate MIU's time-resolved capabilities through high-resolution structure determination of mixed samples, pH-induced viral capsid contraction, and ligand-dependent complex formation. These findings establish MIU as a cost-effective, versatile tool for studying rapid biochemical processes and lay the groundwork for future applications to time-resolved cryo-EM.