Lumazine Synthase was expressed in E.coli and purified by immobilised metal affinity chromatography. Particles were frozen on C-flat R1.2/1.3 grids. Imaging was performed at 200 kX magnification on a JEOL CryoARM 300 equipped with a Direct Electron DE64 detector. Images were collected using SerialEM with image-beam shift to record two shots per hole. The energy-filter slit was not inserted. Images were collected in electron counting mode with two times binning. To correct for some residual fixed-pattern in the images, a post-collection gain-reference estimate was calculated using Relion, and applied using MotionCor2 with whole-frame motion correction.
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