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.

Protein Dynamics and Time Resolved Protein Crystallography at Synchrotron Radiation Sources: Past, Present and Future

“…These classical approaches of TRX via populating intermediate states by protein mutations or chemical trapping, gradually fell out of favor in the past decade. However, the recent development of serial crystallography (SX) at XFELs [99], coupled with advances in sample delivery methods [100] and X-ray detectors technology [101][102][103][104][105], has resulted in a renewed interest in TRX, first at free-electron lasers (TR-SFX) (see review [37]) and more recently at synchrotron sources (TR-SSX) (see Table 1).…”

“…The serial femtosecond crystallography (SFX) technique has increased the number of structures of macromolecules in the PDB with 383 currently deposited as of 1 May 2021, as well as expanded the information that can be obtained from them regarding structural dynamics (see review [37]). The high solvent content and the weak forces that stabilize crystal contacts typically present in macromolecular crystals, have always been a challenge to obtain large and well-diffracting crystals.…”

“…To date, numerous TR-SFX experiments have been carried out successfully on both light-activated and rapid-mixing reactions. Examples of the most relevant and exciting discoveries in the emergent technology of TR-SFX at XFELs have been gathered in a recently published review [37].…”

Approach of Serial Crystallography

“…Serial crystallography is a technique that can overcome the experimental limitations observed in the traditional X-ray technique [6,24,25]. SX uses X-ray free electron laser (XFEL) or synchrotron X-ray, wherein the crystals are exposed to the X-rays only once, thus minimizing radiation damage compared to the traditional X-ray crystallography technique.…”