X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules

Abstract

X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

“…Pump-probe crystallography offers the unique opportunity to capture successive snapshots of light-activated processes to study structural changes at the level of individual atoms (Neutze & Moffat 2012). X-ray free electron lasers (XFELs) and the emerging method of time-resolved serial femtosecond crystallography (TR-SFX) brought exciting new opportunities for structural biologists (Schlichting 2015;Chapman 2019). Recent studies on the photoactive yellow protein (Tenboer et al 2014; Pande et al 2016) proved that TR-SFX is a powerful new tool to study the structural dynamics of proteins.…”

The HARE chip for efficient time-resolved serial synchrotron crystallography

“…This method has experienced a renaissance since the advent of high-brilliance X-ray free-electron laser (XFEL) sources. XFELs triggered the development of serial diffraction data-collection methods because a single high-intensity FEL pulse can lead to the destruction of the crystal under study (Chapman, 2019;Chapman et al, 2011). Serial approaches have the additional advantage of avoiding many of the difficulties traditionally associated with time-resolved studies of single crystals, i.e.…”

“…reaction initiation, radiation damage and signal-to-noise. Taking advantage of the ultrashort timeresolutions accessible at XFELs, many of the initial FEL studies have probed sub-picosecond timescales using liquid-jet delivery systems (Martin-Garcia et al, 2016;Kupitz et al, 2017Kupitz et al, , 2014Lee et al, 2018;Schlichting, 2015;Chapman, 2019;Tenboer et al, 2014;Barends et al, 2015). Liquid jets brought new challenges: collecting thousands of still diffraction patterns mandates high-velocity crystal exchange, which initially resulted in large sample consumption and waste.…”

“…While XFELs are uniquely suited to provide insight into ultrafast dynamics on the femtosecond scale, metastable reaction intermediates of most enzyme mechanisms occur on the microsecond to second time domain and this can conveniently be addressed at third-and fourth-generation synchrotron sources (Schlichting, 2015;Chapman, 2019;Bar-Even et al, 2011;Weinert et al, 2019;Schulz et al, 2018;Mehrabi, Schulz, Dsouza et al, 2019;Mehrabi, Schulz, Agthe et al, 2019). However, a significant problem when utilizing serial approaches for these longer delay times is the inevitable increase in overall data-collection time for a standard pumpdelay-probe experiment, which makes data acquisition at long time delays impractical within a standard 24 hour beam time.…”

Protein phase separation and determinants of in cell crystallization

“…This opens new avenues to explore disease related in vivo crystals as drug target . In cellulo protein crystallization has also gained attention as a new and alternative method to produce high amounts of micro‐ or nano‐sized crystals which can be used to determine the 3D structure of the crystallized protein using either high brilliant X‐ray free electron laser or highly brilliant micro‐focused synchrotron radiation applying serial diffraction data collection . However, despite an increasing number of publications reporting in vivo crystallization, the physicochemical parameters required and the molecular mechanism of in vivo crystallization guiding crystallization in cells are up to date only poorly understood, considering that even conventional, in vitro, protein crystallization till now remains a challenge .…”