X‑ray Emission Spectroscopy as an in\nSitu Diagnostic Tool for X‑ray Crystallography of\nMetalloproteins Using an X‑ray Free-Electron Laser

Serial\nfemtosecond crystallography (SFX) using the ultrashort X-ray\npulses from a X-ray free-electron laser (XFEL) provides a new way\nof collecting structural data at room temperature that allows for\nfollowing the reaction in real time after initiation. XFEL experiments\nare conducted in a shot-by-shot mode as the sample is destroyed and\nreplenished after each X-ray pulse, and therefore, monitoring and\ncontrolling the data quality by using in situ diagnostic\ntools is critical. To study metalloenzymes, we developed the use of\nsimultaneous collection of X-ray diffraction of crystals along with\nX-ray emission spectroscopy (XES) data that is used as a diagnostic\ntool for crystallography, by monitoring the chemical state of the\nmetal catalytic center. We have optimized data analysis methods and\nsample delivery techniques for fast and active feedback to ensure\nthe quality of each batch of samples and the turnover of the catalytic\nreaction caused by reaction triggering methods. Here, we describe\nthis active in situ feedback system using Photosystem\nII as an example that catalyzes the oxidation of H2O to\nO2 at the Mn4CaO5 active site. We\nused the first moments of the Mn Kβ1,3 emission spectra,\nwhich are sensitive to the oxidation state of Mn, as the primary diagnostics.\nThis approach is applicable to different metalloproteins to determine\nthe integrity of samples and follow changes in the chemical states\nof the reaction that can be initiated by light or activated by substrates\nand offers a metric for determining the diffraction images that are\nused for the final data sets.