Abstract
The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.
Highlights
Time-resolved macromolecular crystallography (TRX) unifies structure determination with reaction dynamics
Structures that evolve along the reaction coordinate can be determined with near atomic resolution in concert with the kinetic mechanism using the same set of crystallographic data
This opens the previously unreachable ultrafast time scales to crystallography
Summary
Time-resolved macromolecular crystallography (TRX) unifies structure determination with reaction dynamics. The enormous X-ray pulse intensities at these machines makes it possible to interrogate a tiny crystal with a single X-ray pulse that lasts only femtoseconds This opens the previously unreachable ultrafast time scales to crystallography. The integrated intensities are assembled from hundreds to thousands of observations for each individual reflection gathered from a large number (millions) of diffraction patterns These patterns were collected from trillions of tiny crystals that were injected in serial fashion into the X-ray beam. This approach, called Monte-Carlo integration [11,12], enabled the collection of complete datasets of highly accurate reflection intensities suitable for de novo X-ray structure determination [13] and TRX experiments
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