Abstract
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
Highlights
A full understanding of the function and interactions of biological macromolecules requires a determination of their three-dimensional (3D) atomic structures and the dynamics of the rearrangement of these structures as reactions occur
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet
We describe here a new approach that combines elements of time-resolved crystallography and wide-angle X-ray scattering (WAXS), in which diffraction patterns from diffracting microcrystals flowing in liquid suspension [13, 14] are recorded using femtosecond pulses from an X-ray free-electron laser (FEL)
Summary
A full understanding of the function and interactions of biological macromolecules requires a determination of their three-dimensional (3D) atomic structures and the dynamics of the rearrangement of these structures as reactions occur. Time-resolved measurements require a fast trigger for initiating the reaction in the crystal, which is probed at different time points along the reaction coordinate, with the crystal in different orientations, until complete data sets are obtained. Both reversible (e.g., [7,8]) and irreversible (e.g., [9]). The analysis of conformational changes in large protein complexes by time-resolved Laue crystallography is, hampered by the inherent problem of overlapping reflections, and is normally used only to study reproducible processes that can be repeatedly excited by an external trigger
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