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Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments

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Schlichting,  Ilme
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Shoeman,  Robert L.
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Schirò, G., Woodhouse, J., Weik, M., Schlichting, I., & Shoeman, R. L. (2017). Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments. Journal of Applied Crystallography, 50, 932-939. doi:10.1107/S1600576717006264.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002D-6F6F-A
Abstract
Proteins that change their structure in response to light absorption regulate many functional processes in living cells. Moreover, biotechnological approaches like optogenetics and super-resolution fluorescence microscopy recently triggered the generation of new genetically modified photosensitive proteins. Light-induced structural changes in photosensitive proteins can be studied by time-resolved serial femtosecond crystallography (SFX), an X-ray diffraction technique that allows the determination of macromolecular structures at X-ray free-electron lasers from a large number of nano- to micro-sized crystals. This article describes a simple and efficient system for converting photosensitive proteins into light-induced semi-stationary states by inline laser illumination prior to sample injection with a gas-focused liquid jet and subsequent optical pump–X-ray probe exposure. The simple setup of this device makes it suitable for integration into other liquid injectors (like electro-spinning and electro-kinetic injectors) and potentially also in high-viscosity extruders, provided that embedding microcrystals in viscous media does not alter protein photophysical properties. The functioning of the device is demonstrated with an example of a photoswitchable fluorescent protein pre-illuminated (photoactivated) for time-resolved SFX experiments. The device can be easily adapted for the conversion in time-resolved SFX experiments of other microcrystalline proteins, such as photosystems, phytochromes and rhodopsins.