A Bright Future for Serial Femtosecond Crystallography with XFELs

Abstract

X-ray free electron lasers (XFELs) have potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance and short duration of X-ray pulses. A recently emerged serial femtosecond crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense femtosecond XFEL pulses enable high-resolution room temperature structure determination of difficult to crystallize biological macromolecules, while simultaneously opening up a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs.

The New Era of Microcrystallography

“…Serial femtosecond crystallography (SFX) enables data collection by streaming across the beam, thousands of small, hydrated, randomly oriented protein crystals using a 'one crystal, one shot' approach. 19 The use of XFEL in serial crystallography has enabled structural biologists to probe nano-and micrometer-sized crystals. Figure 4 schematically depicts an SFX experiment using XFEL sources.…”

“…23 Novel technologies related to data management strategies are being continuously developed to handle such large amounts of data. 19 Beamlines with a cross-section in the range of 1-20 μm are known as microfocus beamlines. The increasing availability of dedicated microfocus beamlines has allowed a great expansion of the use of microcrystals for structure determination.…”

A statistical model for improved membrane protein expression using sequence-derived features

“…This example highlights the funnel problem of structural biology, where each stage of the structure pipeline eliminates a large percentage of targets compounding into an overall low rate of success (5). With new and rapidly advancing technologies like cryo-electron microscopy, serial femtosecond crystallography, and micro-electron diffraction, we expect that the latter half of the funnel, structure determination, will increase in success rate (6)(7)(8). However, IMP expression levels will continue to limit targets accessible for study (9).…”

Macromolecular X-ray crystallography: soon to be a road less travelled?

“…The decline in MX PDB depositions may also reflect the end of the era of 'low-hanging fruit' in MX and, as projects become more challenging, more resources are required to generate the same level of output. This latter point can best be observed by the development of serial crystallography at XFELs (Johansson et al, 2017) and now also electron diffraction (for recent reviews, see Clabbers & Abrahams, 2018;Nannenga & Gonen, 2019). These methods have further enabled the collection of diffraction data from submicrometre crystalline samples and, in the case of serial crystallography, have made room-temperature, time-resolved crystallography experiments a reality.…”