Serial Femtosecond Crystallography: A Decade at the Forefront in Structural Biology

Abstract

Exceptionally brilliant, femtosecond‐pulsed X‐ray sources, the X‐ray free‐electron lasers (XFELs), have brought a new way of conducting crystallography by probing nano/micrometre‐sized crystals in a serial fashion. Since the first XFEL, the Linac Coherent Light Source (LCLS), started operation in 2009, the serial femtosecond crystallography (SFX) technique has opened up new exciting opportunities for the determination of static structures as well as the structural dynamics of macromolecules. Here we gathers information from the greatest advances and exciting discoveries in the past 10 years in the emerging technology of SFX at XFELs as well as outlines the frontiers of this technology at upcoming compact pulsed X‐ray sources and its implementation at synchrotron radiation sources.

Key Concepts

  • Reviewed 10 years of successful science at X‐ray free‐electron lasers (XFELs).
  • Highlighted the major key advances of the SFX technique in the areas of sample delivery and data analysis.
  • Highlighted the breakthrough experiments to determine the structure of highly relevant therapeutic targets such as GPCRs.
  • Highlighted the breakthrough experiments to study structural dynamics of macromolecules using the time‐resolved technique.
  • Outlined the frontiers of serial crystallography technology at modern XFELs and compact instruments as well as its implementation at synchrotron radiation sources.

Keywords: X‐ray free‐electron lasers; synchrotron radiation sources; serial crystallography; time‐resolved serial femtosecond crystallography; nano/microcrystals; structural dynamics; enzymatic reactions; light‐activated reactions

Figure 1. The LCLS at SLAC National Laboratory. (a) Aerial overview of the LCLS. Different sections of the LCLS machine are highlighted. (b) Undulator hall at the LCLS. Emma et al., . (c) Schematic layout of the LCLS machine. Electron bunches are generated at the electron gun (RF Gun) and travel downstream passing through three LINACs and two bunch compressor chicanes and two beam diagnostics sections. The XFEL beam is produced by the undulators. XFEL beam and electrons are separated so that electrons are dumped and XFEL beam goes to the near and far experimental halls, NEH and FEH, respectively, for conducting SFX experiments.
Figure 2. Timeline of GPCRs at XFELs. All GPCR structures determined in 10 years of XFELs are shown.
Figure 3. Typical schematic set‐ups of TR‐SFX experiments at XFELs. (a) Set‐up of a time‐resolved mix‐and‐inject experiment in which protein microcrystals are mixed with a substrate in the interaction region of the injector (black boxes) so that the substrate rapidly diffuses into the crystals and binds to the protein (red boxed). Time delays can be probed by varying the sample and buffer flow rates or by placing an expanded region after the constriction. Crystal and substrate are delivered into the FEL beam, and the data set is collected on the detector. (b) Set‐up of a time‐resolved pump–probe experiment in which crystals are injected into the path of a pump laser before being probed after a predetermined time delay (Δt) by the X‐rays. Like in (a), data sets are collected on the detector (not shown).
Figure 4. Examples of time‐resolved XFEL studies of light‐activated proteins. (a) Ultrafast changes in the chromophore of PYP capture by TR‐SFX. As shown in the inset panel, the PCA chromophore remains in a trans configuration (purple structure) after 250 fs, and 3 ps after light excitation the configuration is cis (green structure). The yellow structure represents the dark state. Adapted from Pande et al. (). (b) TR‐SFX has provided great success in revealing the water‐splitting mechanism by PSII including structural changes upon OO bond formation in the manganese cluster (inset panel 1; inset panel 2). Adapted from Suga et al. () and Kern et al. (). (c) TR‐SFX of bacteriorhodopsin captured the primary photochemical events of the trans to cis isomerisation of the retinal at short time delays (Δt = fs to ps). Adapted from Nogly et al. (). (d) Ultrafast motions in CO myoglobin upon ligand dissociation by TR‐SFX. Inset panel shows the different electron density maps of the bound CO (red map) and photodissociated CO (green map), doming of the heme, out‐of‐plane movement of the iron and concomitant movement of His93 away from the heme. Adapted from Barends et al. ().
Figure 5. Examples of mix‐and‐inject TRS‐SFX at XFELs. (A) Inactivation of the antibiotic CEF by BlaC was watched by MISC. Simulated annealing ‘omit’ difference density maps of the active site structures (a–e) during the reaction at time points between 30 ms and 2 s upon mixing are shown. All species of the kinetic reaction were captured. Olmos et al. (). CC BY 4.0. (B) MISC experiment of a riboswitch RNA during its reaction with adenosine followed by a 10‐s delay. A closer view of the active site of apo1, apo2, intermediate (IB) and ligand‐bound states is illustrated in the box. Lower panels show the key residues in the ligand‐binding pockets of all protein states. Adapted from Stagno et al. (). (C) Snapshot of the PR oxygen intermediate in the catalytic reaction of CcO followed by MISC. The inset panel shows the electron density maps of the binuclear centre of the PR intermediate in the reduced state of CcO (left) and the PR intermediate with a ferryl oxygen coordinated to the haem a3 and a hydroxide coordinated to CuB (right). Adapted from Ishigami et al. ().
Figure 6. Layout of the components needed for a compact X‐ray source. Electron bunches generated by the photoinjector are accelerated and compressed by the linacs and compression chicanes devices, respectively. The interaction point, where electrons and laser collide to produce the X‐rays by the ICS phenomenon, is also highlighted. The total length from the photoinjector to ICS interaction point is about 10 m. Components in the experimental hutch (beam shaping and diagnostics, sample chamber and detector) are also illustrated.
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Further Reading

Chapman HN and Fromme P (2017) Structure determination based on continuous diffraction from macromolecular crystals. Current Opinion in Structural Biology 45: 170–177.

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Zatsepin NA, Li C, Colasurd P and Nannenga BL (2019) The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals. Current Opinion in Structural Biology 58: 286–293.

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Fromme, Petra, Graves, William S, and Martin‐Garcia, Jose M(May 2020) Serial Femtosecond Crystallography: A Decade at the Forefront in Structural Biology. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028964]