Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

X-ray fluorescence holography of biological metal sites: Application to myoglobin.

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules.