Droplet microfluidics for time-resolved serial crystallography

Stubbs, J.; Hornsey, T.; Hanrahan, N.; Blay-Esteban, L.; Bolton, R.; Maly, M.; Basu, S.; Orlans, J.; de Sanctis, D.; Shim, J.-u.; Shaw Stewart, P. D.; Orville, A. M.; Tews, I.; West, J.

doi:10.1101/2024.01.12.575388

bioRxiv · 10.1101/2024.01.12.575388

Droplet microfluidics for time-resolved serial crystallography

Stubbs, J.·Hornsey, T.·Hanrahan, N.·Blay-Esteban, L.·Bolton, R.·Maly, M.·Basu, S.·Orlans, J.·de Sanctis, D.·Shim, J.-u.·Shaw Stewart, P. D.·Orville, A. M.·Tews, I.·West, J.

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Abstract

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here we report the use of droplet miniaturisation for the controlled production of uniform crystals, providing an avenue for controlled diffusion and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3-{micro}m lysozyme crystals and 2-{micro}m crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 milliseconds were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be used to advance time-resolved serial crystallography.

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Stubbs, J., Hornsey, T., Hanrahan, N., Blay-Esteban, L., Bolton, R., Maly, M., Basu, S., Orlans, J., de Sanctis, D., Shim, J.-u., Shaw Stewart, P. D., Orville, A. M., Tews, I., West, J.. 2024-01-13. Droplet microfluidics for time-resolved serial crystallography. https://doi.org/10.1101/2024.01.12.575388

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