Serial femtosecond crystallography: A revolution in structural biology

Abstract

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein.

The Linac Coherent Light Source: Recent Developments and Future Plans

“…Serial femtosecond crystallography (SFX) at XFEL sources [1,[15][16][17] has revolutionized the ability to determine macromolecular structures that are inaccessible by synchrotron sources. Furthermore, time-resolved SFX maps the conformational dynamics that determine biological function, and enables studies at room temperature (near physiological conditions).…”

The New Era of Microcrystallography

“…SFX experiments generate large datasets comprising of snapshots, with each snapshot capturing Bragg diffraction of single crystals in random orientations just before their destruction. 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.…”

Analysis of XFEL serial diffraction data from individual crystalline fibrils

“…Various software packages have been developed for this processing chain (Barty et al, 2014;White et al, 2012White et al, , 2016White et al, , 2017Sauter et al, 2013;Kabsch, 2014;Liu & Spence, 2016). The success of this approach is shown by approximately 100 entries in the PDB of structures determined by SFX (Martin-Garcia et al, 2016).…”

“…In the context of structural biology, the most successful application of XFELs has been through serial femtosecond crystallography (SFX) (Chapman et al, 2011;Spence et al, 2012;Chapman, 2015;Fromme, 2015;Schlichting, 2015;Martin-Garcia et al, 2016). XFELs deliver a coherent femtosecond-duration hard X-ray pulse to a molecular specimen, achieving high-angle elastic scattering before inelastic processes destroy the molecule (Neutze et al, 2000).…”

“…Such crystals are easier to obtain than those of at least tens of micrometres across that are required by synchrotron sources. Crystals are usually injected into the XFEL pulse path through a nozzle as a suspension in a liquid jet Nelson et al, 2016;Oberthuer et al, 2017) oriented perpendicular to the incident X-ray path, although other sample-delivery methods are also in use Chavas et al, 2015;Sierra et al, 2015;Roedig et al, 2016;Martin-Garcia et al, 2016;Fuller et al, 2017). The maximum pulse repetition rate at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory is 120 Hz, and diffraction patterns are usually recorded at this rate (Bostedt et al, 2016).…”