Serial femtosecond crystallography: the first five years

Abstract

Protein crystallography using synchrotron radiation sources has had a tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radiation damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures. With the advent of free-electron lasers (FELs) capable of delivering extremely intense femtosecond X-ray pulses, this situation appears to be remedied, allowing the structure determination of undamaged macromolecules using either macroscopic or microscopic crystals. The latter are exposed to the FEL beam in random orientations and their diffraction data are collected at cryogenic or room temperature in a serial fashion, since each crystal is destroyed upon a single exposure. The new approaches required for crystal growth and delivery, and for diffraction data analysis, including de novo phasing, are reviewed. The opportunities and challenges of SFX are described, including applications such as time-resolved measurements and the analysis of radiation damage-prone systems.

Keywords: FELs; SFX; X-ray lasers; microcrystals; radiation damage; serial femtosecond crystallography; time-resolved crystallography.

Figure 1

A simplified scheme of a protein crystallization phase diagram. In addition to precipitant concentration, the pH, temperature or additive concentrations are also important adjustable parameters. Crystallization approaches to reach the nucleation and metastable zones are indicated.

Figure 2

Data collection approaches. (a) Randomly oriented micro- and nanocrystals can be delivered into the X-ray interaction region using various forms of liquid jets. (b) Large cryo-cooled crystals can be mounted in loops. Serial quasi-rotation data can be collected using a goniometer setup, translating the crystal stepwise by a value Δx,y, an experimentally determined damage zone value, while rotating it by Δϕ, a fraction of its experimentally determined mosacity. Other approaches such as chips can also be used.