Negative-stain single-particle EM was used for direct visualization of the IMAC elution fraction of a purified BcsHisHFL variant carrying the cis-filamentation–disruptive L339D-L343D double mutation upon coexpression with BcsD (BcsHisHFL_L339D-L343D−BcsD). Briefly, 5 μl of IMAC-eluted sample (concentrations, ~0.01 to 0.02 mg/ml) was spotted on glow-discharged carbon-coated copper grids (Agar Scientific). After 60-second incubation, the extra liquid was blotted off, and the grids were passed sequentially through three drops of 2% (w/v) uranyl acetate solution, with 30-second incubation in the last drop before blotting and air drying. Micrographs were taken on a Thermo Fisher Scientific FEI Tecnai F20 electron microscope operated at an accelerating voltage of 200 kV and equipped with a field emission gun and an Eagle 4k × 4k charge-coupled device (CCD) camera. Micrographs were collected with a nominal defocus range of −1.5 to −3 μm and a low dose of ~30 electrons/Å at a pixel size of 1.835 Å2. Micrograph contrast transfer function (CTF) correction was performed with Gctf (47 (link)) through the cryoSPARC v3 interface (48 (link)), and autopicking and class averaging were performed with cryoSPARC’s “Blob picker” and 2D classification tools, respectively.