X-ray Emission Spectroscopy as an in Situ Diagnostic Tool for X-ray Crystallography of Metalloproteins Using an X-ray Free-Electron Laser

Abstract

Serial femtosecond crystallography (SFX) using the ultrashort X-ray pulses from a X-ray free-electron laser (XFEL) provides a new way of collecting structural data at room temperature that allows for following the reaction in real time after initiation. XFEL experiments are conducted in a shot-by-shot mode as the sample is destroyed and replenished after each X-ray pulse, and therefore, monitoring and controlling the data quality by using in situ diagnostic tools is critical. To study metalloenzymes, we developed the use of simultaneous collection of X-ray diffraction of crystals along with X-ray emission spectroscopy (XES) data that is used as a diagnostic tool for crystallography, by monitoring the chemical state of the metal catalytic center. We have optimized data analysis methods and sample delivery techniques for fast and active feedback to ensure the quality of each batch of samples and the turnover of the catalytic reaction caused by reaction triggering methods. Here, we describe this active in situ feedback system using Photosystem II as an example that catalyzes the oxidation of H₂O to O₂ at the Mn₄CaO₅ active site. We used the first moments of the Mn Kβ_1,3 emission spectra, which are sensitive to the oxidation state of Mn, as the primary diagnostics. This approach is applicable to different metalloproteins to determine the integrity of samples and follow changes in the chemical states of the reaction that can be initiated by light or activated by substrates and offers a metric for determining the diffraction images that are used for the final data sets.

Fig. 1

Schematic for simultaneous XRD and XES measurements using either a jet injector or the Drop-on-Tape (DOT) method giving both diffraction images and XES spectrum. This set-up utilizes a von Hamos spectrometer for XES data collection, which enables the detection of the entire Kβ_1,3 spectra in an energy dispersive mode on a position sensitive 2D detector on a shot-by-shot basis. Upper left: energy diagram of Kα and Kβ_1,3 transitions.

Fig. 2

(left) Total detector image of the dark state (0-Flash, 0F) collected during experiment 1. Vertical lines indicating ROI, and vertical dashed lines indicating slices for background subtraction and comparison region for hit finder. The image is the sum of all XES images counted as hits (totally 22,606, with 177,639 detected emission photons). (right) Average hit/indexing rates of different sample batches from experiment 1, estimated using the spectroscopic hit finder (red) and indexing rates from crystallography (blue).

Fig. 3

Kβ_1,3 emission spectra of the 0F and 2F flash states from experiment 1, as well as the 0F-2F difference spectrum (enlarged by a factor of 5). The spectra were area normalized inside the adopted energy interval. The smoothed spectra have been constructed by first binning the raw spectra to 0.75 eV resolution and then using a cubic spline to construct smoothed spectra of 0.01 eV resolution. The 0F data contains 22,606 shots and totally 177,639 emission photons, while 2F contains 67,219 shots and 590,156 emission photons.

Fig. 4

(left) First moment variance of all measured flash states with respect to total number of detected Kβ_1,3 emission photons, also indicating the 0F and 2F data used in Fig. 3. Included is also a power fit, yielding close to a square-root dependence and a R² value of 0.996. (right) 0F and 2F first moments as a function of number of photons, illustrated for data collected during experiment 1. Indicated is also the estimated first moment variance (dashed lines, in blue for 0F and green 2F) using the power fit with parameters from the left panel, as centered around final first moments (dotted-dashed lines in red).

Fig. 5

0F_est first moments and Mn(II) levels of different sample batches from experiment 2. Horizontal dashed line is the 0F first moment, which has negligible Mn(II), and higher percentages are constructed by considering the Mn(II) first moment from MnCl₂ reference measurements. XES estimates include standard deviation for photon count.

Fig. 6

First moment progressions of three anomalous sample batches from experiment 2, as compared to the averages of the good batches. Standard deviations calculated by random sampling of data sets (200 iterations).