Abstract
Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.
Highlights
Since their first operation in 2009, X-ray free-electron lasers (XFELs) have opened up new, previously unreachable possibilities for structural biology research in the domains of time resolution and radiation exposure
The first crystal injector to be used at an XFEL was the gas dynamic virtual focusing nozzle (GDVN; DePonte et al, 2008; Ganan-Calvo, 1998), which serves as a workhorse for SFX sample delivery (Fig. 2a)
The advent of XFELs has recently brought about remarkable developments in sample-delivery methods for MX experiments
Summary
Since their first operation in 2009, X-ray free-electron lasers (XFELs) have opened up new, previously unreachable possibilities for structural biology research in the domains of time resolution and radiation exposure. To most efficiently collect a data set, fresh sample must be brought into the beam between X-ray pulses, requiring a sample-exchange rate that matches the repetition rate of the XFEL source. The crosstalk between XFEL and synchrotron facilities has brought about a new wave of innovation in macromolecular crystallography (MX) sample delivery, data collection and data analysis, expanding the possibilities in structural biology research. Exposing larger crystals enables the collection of stronger diffraction and higher resolution data, an approach that is well suited for detailed structural investigations of the active sites of redox-active enzymes that are highly susceptible to X-ray-induced photoreduction at the synchrotron. The use of larger crystals with goniometer setups can dramatically improve the efficiency of the SFX experiment as they may be exposed in multiple volumes and may be rotated and translated between X-ray pulses to boost the completeness
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