Fixed-target time-resolved crystallography at XFELs: the scourge of light contamination but reduced sample consumption

Author:

Gotthard Guillaume,Flores-Ibarra Andrea,Carrillo Melissa,Kepa Michal W,Mason Thomas J,Stegmann Dennis P.,Olasz Bence,Pachota Magdalena,Dworkowski Florian,Ozerov Dmitry,Pedrini Bill F.,Padeste Celestino,Beale John H,Nogly PrzemyslawORCID

Abstract

AbstractX-ray free electron laser (XFEL) light sources have allowed for the rapid growth of time-resolved structural experiments, which provide crucial information on the function of biological machines and their mechanisms. We set out to commission the SwissMX fixed-target sample delivery system at the SwissFEL Cristallina experimental station using the PSI developed MISP-chip for pump-probe time-resolved experiments. To characterise the system, we used the light-sensitive protein crystals of the Light-Oxygen-Voltage domain 1 (LOV1) fromChlamydomonas reinhardtii. Using different experimental settings, the adjacent-well light contamination was carefully assessed, indicating that it is crucial to control the light scattering from solid supports otherwise significant contamination can occur. However, our results show that, after the initial experiments and parameter refinement, the opaque MISP-chips are suitable for pump-probing a light-sensitive protein. This crystallographic experiment also probed the sub-millisecond structural dynamics of the LOV1 and indicated that at Δt=10 μs the covalent thioether bond is already established between the reactive Cys57 and FMN cofactor. This experiment validated the crystals to be suitable for in-depth follow up studies of the still poorly understood signal transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared to the most successful system used at XFEL, the high-viscosity extruder. This development creates the prospect of an exciting increase in XFEL project throughput for the field.

Publisher

Cold Spring Harbor Laboratory

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