Abstract
The ultrabright and ultrashort pulses produced at X-ray free electron lasers (XFELs) has enabled studies of crystallized molecular machines at work under ‘native’ conditions at room temperature by the so-called time-resolved serial femtosecond crystallography (TR-SFX) technique. Since early TR-SFX experiments were conducted at XFELs, it has been largely reported in the literature that time-resolved X-ray experiments at synchrotrons are no longer feasible or are impractical due to the severe technical limitations of these radiation sources. The transfer of the serial crystallography approach to newest synchrotrons upgraded for higher flux density and with beamlines using sophisticated focusing optics, submicron beam diameters and fast low-noise photon-counting detectors offers a way to overcome these difficulties opening new and exciting possibilities. In fact, there is an increasing amount of publications reporting new findings in structural dynamics of protein macromolecules by using time resolved crystallography from microcrystals at synchrotron sources. This review gathers information to provide an overview of the recent work and the advances made in this filed in the past years, as well as outlines future perspectives at the next generation of synchrotron sources and the upcoming compact pulsed X-ray sources.
Highlights
The ultrabright and ultrashort pulses produced at X-ray free electron lasers (XFELs) has enabled studies of crystallized molecular machines at work under ‘native’ conditions at room temperature by the so-called time-resolved serial femtosecond crystallography (TR-SFX) technique
Temporal resolution regimes, opening the door to observing conformationally dynamic proteins in action at atomic scale. While this technique continues to improve, it has two main limitations: (1) the time resolution currently achievable with even the best synchrotron radiation source is limited by the X-ray pulse length of approximately 100 ps; and (2) it requires a rapid reaction initiation that quickly spreads uniformly across all molecules in the crystals and with high efficiency, and this represents a clear challenge with large crystals
This study clearly demonstrates the high applicability of the liquid application method for time-resolved analysis (LAMA) method for time-resolved SSX (TR-SSX) studies in the millisecond to high seconds time regime
Summary
“Watching” biological macromolecules in action at atomic resolution rather than just being limited to 3D-dimensional static pictures, has been the holy grail for structural biologists since the first high-resolution structure of an enzyme was solved [1]. The ideal bandwidth to fully record reflections with maximum SNR is probably slightly larger than that required to match the crystal mosaicity Despite this disadvantage, the technique of Laue crystallography has rapidly evolved over the past 25 years with developments in cryo-technology, tunable lasers, increased computing power and vastly improved X-ray detectors, as well as synchrotron radiation itself, which has allowed the technique to move from the millisecond [5] to the picosecond temporal resolution regimes [18]. It has recently been demonstrated that the binding of an antibiotic to an enzyme can be studied by collecting data at room temperature using time-resolved SFX at LCLS [31,32] Such studies have remained largely elusive at synchrotron sources because of X-ray radiation damage, the need for growing large single crystals, challenges with crystal replenishment, and the difficulty in initiating reactions uniformly in macroscopic crystals. It highlights the most recent advances towards developing the first mix-and-inject time-resolved experiments at synchrotrons
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