Abstract
Structural information on protein dynamics is a critical factor in fully understanding the protein functions. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) is a recently established technique for visualizing the structural changes or reactions in proteins that are at work with high spatial and temporal resolution. In the pump-probe method, protein microcrystals are continuously delivered from an injector and exposed to an X-ray free-electron laser (XFEL) pulse after a trigger to initiate a reaction, such as light, chemicals, temperature, and electric field, which affords the structural snapshots of intermediates that occur in the protein. We are in the process of developing the device and techniques for pump-probe TR-SFX while using XFEL produced at SPring-8 Angstrom Compact Free-Electron Laser (SACLA). In this paper, we described our current development details and data collection strategies for the optical pump X-ray probe TR-SFX experiment at SACLA and then reported the techniques of in crystallo TR spectroscopy, which is useful in clarifying the nature of reaction that takes place in crystals in advance.
Highlights
X-ray crystallography is a powerful tool for determining the protein structure at high spatial resolution
We describe in crystallo TR spectroscopy to assess the structural dynamics in the crystalline phase, which should be performed before conducting TR-SFX
A sample is ejected from an which yields high quality microcrystals in lipidic cubic phase (LCP), with the Nd:YAG laser at a delay time (Δt) of 250 μs injector that was mounted on a motorized manipulator and it is delivered to an X-ray free-electron laser (XFEL) focal point
Summary
X-ray crystallography is a powerful tool for determining the protein structure at high spatial resolution. X-ray free-electron lasers (XFELs) produce intense, ultrashort pulses; as such, XFELs have enabled room-temperature protein structure determination via diffraction patterns from microcrystals before any manifestation of radiation damage [2]. The project established the foundation of devices and techniques for SFX at SACLA for the first five years This foundation has led to the development of a setup for pump-probe time-resolved serial femtosecond crystallography (TR-SFX), which uses a pump laser pulse to trigger reactions.
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