From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons

Abstract

Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump–probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.

The nucleating agent crystallophore induces instant protein crystallization

“…As a result, TR-SX allows to obtain structural dynamics information and observe biological macromolecules in action by capturing transient intermediates along a biological pathway. 3–6…”

“…As a result, TR-SX allows to obtain structural dynamics information and observe biological macromolecules in action by capturing transient intermediates along a biological pathway. [3][4][5][6] From an experimental point of view, serial crystallography brings new constraints on crystal preparation as it intrinsically requires a large amount of samples to make sure to collect a complete diffraction data set. Moreover optimal time-resolved experiments require crystalline samples with a narrow size distribution in order to ensure a uniform triggering of the reaction under study through the entire crystal volume.…”