Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser

Abstract

X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.

FIG. 1

(Color) Experimental setup. Randomly oriented lysozyme nanocrystals (blue) were introduced into the focus of the x-ray beam (yellow) by a gas-focused water jet (gray) (Ref. 18). Diffraction data (white spots) were collected using a pair of pnCCD detectors (black) (Refs. and 17), set at different vertical distances with respect to the beam to cover high scattering angles.

FIG. 2

(Color) Virtual powder diffraction data collected on a pair of pnCCD detectors and comparison of intensities for the different pulse durations. (a) Section of the virtual powder pattern (70 fs SFX data) calculated for the upper pnCCD detector. Scattering of the liquid jet column containing the nanocrystals produces the vertical streak shown in the lower-left corner. (b) Section of the lower detector. Intensities are scaled by a factor of 3 for better visualization. (c) Transformation of the upper (blue) and lower (red) detector data into a 1-dimensional intensity plot. (d) Radiation damage at different pulse durations analyzed by comparing scattered intensities for different resolution bins. Intensities were averaged by moving a sliding window over the 1-dimensional powder patterns. All intensities were normalized in the low resolution range (0.002, 0.006 Å⁻²) and compared to the intensities of the 70-fs data. Very low resolution data is affected by stray light from the liquid [gray vertical dots in (a)] and were therefore omitted from the analysis.

FIG. 3

(Color) Comparison of powder pattern intensities for different pulse durations and diffraction pattern intensities. Diffraction intensities of all pulse lengths and scattered intensity classes were treated as in Fig. 2(d). Intensities of all classes (different layers) and all pulse lengths are compared to the 70 fs data set of the lowest intensity class. The color encodes relative intensity.