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| Title | `Mix-it-up': accessible time-resolved cryo-EM on the millisecond timescale. |
|---|---|
| Journal, issue, pages | IUCrJ, Vol. 12, Issue Pt 6, Page 710-724, Year 2025 |
| Publish date | Nov 1, 2025 |
 Authors | Lauren Alexandrescu / William Lessin / Gabriel C Lander /  |
| PubMed Abstract | Biological reactions often involve macromolecules that undergo substrate-induced conformational changes in under a second, yet capturing these transient states remains challenging. While high- ...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 ms. 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. |
 External links | IUCrJ / PubMed:41140236 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 2.9 - 6.9 Å |
| Structure data |  EMDB-70747: Mouse heavy-chain apoferritin from mixed sample prepared with Mix-it-up  EMDB-70748: Rabbit muscle aldolase from mixed sample prepared with Mix-it-up  EMDB-70749: Cowpea chlorotic mottle virus in the contracted state  EMDB-70750: Cowpea chlorotic mottle virus in the expanded state  EMDB-70751: Cowpea chlorotic mottle virus in contracted state 700 ms after pH shift triggered using Mix-it-up  EMDB-70752: E.coli GroEL in presence of GroES and in absence of ATP  EMDB-70753: E.coli GroEL in presence of GroES 300ms after addition of ATP  EMDB-70754: E.coli GroEL/ES in the bullet conformation 300ms after addition of ATP |
| Source |
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Mus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
Cowpea chlorotic mottle virus
Escherichia coli (E. coli)