Automated grid-based XFEL diffraction studies of single macromolecular crystals

Abstract

A major challenge of structural investigations of metalloproteins at synchrotrons is the damaging effects of radiation exposure. Even small X-ray doses can reduce or initiate reactions at metal centers modifying the active site. For example, in-situ visible absorption spectroscopy measurements have demonstrated that the heme/copper active site in oxidized ba3 cytochrome oxidase (ba3) is compromised during a single X-ray diffraction exposure. The use of ultrashort X-ray pulses at LCLS provides a means to measure high resolution diffraction before these damage processes occur. To this end, experiments were conducted at LCLS using large multiple crystals (> 50 µm) of ba3, hydrogenase and myoglobin. Crystals were mounted in `grids' or loops and flash frozen. The grids hold up to 75 crystals in known locations and are compatible with the Stanford Automounter used to exchange them. Following a semi-automated grid alignment procedure, a fully automated routine was used to position each crystal and collect a series of diffraction images and the Blu-Ice/DCS control system that coordinated with the LCLS EPICS system and XPP DAQ software. Single femtosecond X-ray pulses produced a `damage free' still diffraction image from each crystal. To provide additional information about crystal orientation, a series of pseudo-oscillation images were collected +/- 5.5 degrees spanning the orientation of the still image. For each one degree oscillation image the crystal was exposed to 120 attenuated X-ray pulses. A hard X-ray spectrometer was used to measure the energy spectrum of each individual X-ray pulse. The details and results of these experiments will be presented.