Methods development for diffraction and spectroscopy studies of metalloenzymes at X-ray free-electron lasers

Abstract

X-ray free-electron lasers (XFELs) open up new possibilities for X-ray crystallographic and spectroscopic studies of radiation-sensitive biological samples under close to physiological conditions. To facilitate these new X-ray sources, tailored experimental methods and data-processing protocols have to be developed. The highly radiation-sensitive photosystem II (PSII) protein complex is a prime target for XFEL experiments aiming to study the mechanism of light-induced water oxidation taking place at a Mn cluster in this complex. We developed a set of tools for the study of PSII at XFELs, including a new liquid jet based on electrofocusing, an energy dispersive von Hamos X-ray emission spectrometer for the hard X-ray range and a high-throughput soft X-ray spectrometer based on a reflection zone plate. While our immediate focus is on PSII, the methods we describe here are applicable to a wide range of metalloenzymes. These experimental developments were complemented by a new software suite, cctbx.xfel. This software suite allows for near-real-time monitoring of the experimental parameters and detector signals and the detailed analysis of the diffraction and spectroscopy data collected by us at the Linac Coherent Light Source, taking into account the specific characteristics of data measured at an XFEL.

Keywords: X-ray crystallography; X-ray emission spectroscopy; X-ray free-electron laser; metalloenzymes; photosystem II; water oxidation.

Figure 1.

Experimental set-up for XRD and X-ray spectroscopy at LCLS. (a) Schematic of the set-up for hard X-ray XRD and XES at the LCLS. Sample is injected from a sample reservoir as a jet using electorspinning for focusing of the jet. The von Hamos XES spectrometer is located at a 90° angle with the X-ray beam, the XES position-sensitive detector (PSD) is located underneath the interaction region. The XRD detector is located in the forward-scattering direction. An optical laser can be used for time-resolved pump–probe studies (modified after [24]). (b) Energy-level diagrams for Mn for K-edge (left panel) and L-edge (right panel) transitions; X-ray absorption processes are shown by red arrows, X-ray emission transitions by blue arrows. (c) Photograph of the set-up installed in the CXI chamber, X-ray beam direction is from right to left. (d) Schematic of the zone plate spectrometer set-up used at the SXR instrument. The inset shows images from the CCD detector recorded at incident X-ray energies below and above the Mn L-edge (640 eV), showing the defocused O Kα signal and the focused Mn Lα signal (modified after [25]). In the inset, Mn-L-edge spectra of Mn^IICl₂ obtained either at the synchrotron in transmission mode (trans. blue) or at the LCLS using partial fluorescence yield (PFY, red) are shown together with calculated Mn-L-edge PFY (pink) and X-ray absorption (XAS, violet) spectra of Mn²⁺ using the multiplet approach. The slight differences between transmission and PFY spectra can be explained by state-dependent fluorescence yield, see [25] for details.

Figure 2.

XRD data processing: (a) online monitoring screen capture image, showing the hit finder result (number of Bragg spots, blue, bottom), the hit rate (green, middle) and the XES signal symmetry (red, top) over time for a thermolysin XRD run at CXI. (b) Detailed view of a diffraction pattern from thermolysin recorded at CXI, showing characteristics of the diffraction spots (spots often extend only over a few pixels and the shape of neighbouring spots can be very different). The expected spot extent using a three-parameter model (lower energy, upper energy and mosaic spread) of the spot shape is shown as red dots together with the Miller indices (red numbers) and the energy limits (black numbers) to fulfil the Bragg condition for each of the spots (adapted from [32]). (c) Diffraction image from thermolysin microcrystals recorded at CXI with two lattices present, showing different resolution for the two lattices (blue ring, 4.20 Å; red ring, 2.35 Å) and the predicted spot positions for both lattices (blue and red circles). (Adapted from [32].)