Cryo-EM Imaging of Carboxysome

Full-length CthACC was diluted to 0.01 mg ml⁻¹ in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp69 (link)70 (link). Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted.