Abstract
With recent developments in X-ray sources, instrumentation and data-analysis tools, time-resolved crystallographic experiments, which were originally the preserve of a few expert groups, are becoming simpler and can be carried out at more radiation sources, and are thus increasingly accessible to a growing user base. However, these experiments are just that: discrete experiments, not just `data collections'. As such, careful planning and consideration of potential pitfalls is required to enable a successful experiment. Here, some of the key factors that should be considered during the planning and execution of a time-resolved structural study are outlined, with a particular focus on synchrotron-based experiments.
Highlights
Macromolecular X-ray crystallography has become nearubiquitous at synchrotrons, which often house multiple heavily automated beamlines dedicated to macromolecular crystallography, permitting routine in-house and even remote data collection without extensive user training
Serial crystallography approaches at both X-ray free-electron lasers (XFELs) and synchrotrons generally encompass four types of sampledelivery methods, which include liquid-injection, microfluidics, fixed-target and hybrid approaches; these have recently been reviewed in detail (Martiel et al, 2019; Grunbein & Nass Kovacs, 2019)
gas dynamic virtual focusing nozzle (GDVN) injectors are well suited to XFEL experiments but are less ideal for synchrotron measurements, especially on monochromatic macromolecular crystallography (MX) beamlines
Summary
Macromolecular X-ray crystallography has become nearubiquitous at synchrotrons, which often house multiple heavily automated beamlines dedicated to macromolecular crystallography, permitting routine in-house and even remote data collection without extensive user training. D78, 14–29 research papers unravelling of biochemical mechanisms of proteins, for example via mutation studies, substrate analogues or freezetrapping techniques (Moffat, 2001) While these classical X-ray crystallographic approaches have been extremely powerful in obtaining insight into equilibrium-state structures, structures obtained from mutants, or with substrate analogues or inhibitors, have the potential to reflect artefacts that only exist in the mutant or particular ligand complex, when trying to probe a reaction mechanism by determining the structure of reaction intermediate-like states. These can go unnoticed when follow-up studies with other techniques or on wild-type proteins are not carried out (Moffat, 2001).
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