Droplet microfluidic and microcrystal strategies for time-resolved serial crystallography of enzymes at synchrotron and XFEL sources

Abstract

Observing transient structural intermediates remains a central challenge in enzymology. Little is known about these despite their pivotal role in catalytic function. Time-resolved serial crystallography at synchrotron and X-ray free electron laser (XFEL) sources offers a promising avenue to capture these dynamic events. However, experimental success hinges on rigorous control of crystallisation, ligand delivery, and integration with beamline infrastructure. This thesis explores the use of droplet microfluidic and microcrystal strategies to address these bottlenecks and establish pipelines for future time-resolved studies.

A high-throughput crystallisation system was developed to generate uniform microcrystals within discrete aqueous droplets, leveraging a seeding strategy to overcome the low probability of nucleation at diminishing volumes. In parallel, a droplet micromixing device was engineered to initiate ligand-triggered reactions on millisecond timescales by exploiting convection within droplets as a means for rapidly mixing microcrystals with ligands. Flow parameters and mixing efficiency were characterised, followed by iterative design and fabrication of a X-ray transmissible device suitable for deployment at a synchrotron beamline.

Arabidopsis thaliana Pyridoxal 5'-phosphate synthase subunit 1.3 (AtPdx1.3) microcrystal slurries were validated for time-resolved studies using static serial femtosecond crystallography (SFX) at the SPring-8 angstrom compact free electron laser (SACLA). High-resolution radiation damage-free structures of apo and ligand-bound AtPdx1.3 were obtained at room temperature, representing the first XFEL structures of this enzyme. Diffraction from 20 μm crystals yielded resolution comparable to or better than previous larger crystals at cryogenic temperatures. Notably, only minimal structural differences were observed relative to cryotrapped structures, indicating strong conformational consistency. Soaking protocols enabled rapid ligand incorporation, capturing R5P, PLP and the crucial I320 intermediate within 15 minutes. These result establish robust workflows for intermediate state trapping and future dynamic studies. Taken together, the platforms developed in this thesis represent a significant step towards realising dynamic structural studies of enzymes at synchrotron and XFEL sources.

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Identifiers

ORCID for Jack Robert Stubbs: orcid.org/0000-0002-3788-1687

ORCID for Ivo Tews: orcid.org/0000-0002-4704-1139

ORCID for Jonathan West: orcid.org/0000-0002-5709-6790

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