Continuous Rotation Electron Diffraction of Lysozyme Microcrystals

Lysozyme microcrystals and EM samples for diffraction were prepared as described previously^{4 (link)}. All electron diffraction was performed with intensity less than 0.01 e⁻/Ų on a TEM operated at 200 kV, equipped with an FEG, and data were collected with 4k × 4k TVIPS TemCam-F416 CMOS camera in rolling shutter mode (15.6 μm pixel size). For continuously rotating diffraction data the stage of the microscope was set to rotate at 0.09° s⁻¹ using the microscope’s standard hardware and software. Crystals were located on the grid by searching in over-focused diffraction mode. When a crystal was found, an initial diffraction pattern was recorded to judge the quality of that particular crystal. If the crystal showed strong and sharp diffraction²⁰ , the beam was blanked and the rotation of the stage was started. Once the stage began its rotation and had achieved a constant rate, the beam was unblanked and diffraction data were recorded at a constant frame rate of 1 frame per 4 s (0.36°/frame) using the camera’s rolling shutter mode. Data sets of approximately 44° were collected for each crystal (total dose <5e⁻/Ų, see supplementary results for dose rate). For the radiation damage assessment, the stage was set at 0° and oscillated back and forth between −1° and 1° during the course of the continuous dosage experiment.