Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.

This article unravels reaction conditions governing the formation of polymorphic structures in solution down to the single particle level applying, for instance, unprecedented real-time serial crystallography measurements during a synthesis process.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray Free Electron Lasers (XFELs) and, more recently, synchrotron sources. Whereas the short pulse duration at XFELs allows for experiments with femtosecond time resolution, the time resolution for serial synchrotron crystallography experiments has been limited to millisecond time scales with monochromatic beams. Using the polychromatic, "pink", beam increases the photon flux by more than two orders of magnitude.Using a setup optimized for very low scattering background we collected polychromatic datasets from four different protein samples at the BioCARS instrument at the APS. Complete datasets were obtained by merging pink beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal. In contrast to serial crystallography experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the high potential of this method for studying irreversible enzyme reactions at sub-microsecond timescales using high-brightness X-ray facilities.