Imaging filter

Data was collected on a Titan Krios 300kV electron microscope (ThermoFisher) equipped with a field emission gun, imaging filter (Gatan, Pleasanton, U.S.) (slit width 20 eV) and K2 or K3 direct electron detector (Gatan). To generate an overview of each grid, grid montages were collected at 135x magnification using SerialEM (Mastronarde, 2005 (link)). Cyanobacteria data was collected with UCSF Tomo (Zheng et al., 2007 (link)) at 2° increments between +60° and −60°. Data was collected at a defocus of −8 µm, total accumulated dose of ~140 e^- / Ų and pixel size of 3.38 Å. Yeast tomograms were collected using SerialEM between +60° and −60° at 2° increments with a defocus of −8 µm, total accumulated dose of ~120 e^- / Ų and pixel size of 4.57 Å. Tomogram reconstruction and subtomogram averaging was performed according to Weiss et al. (2019) (link). Briefly, tomograms were reconstructed using the IMOD package (Kremer et al., 1996 (link)) and septal junction subtomogram averaging was performed using PEET (Nicastro et al., 2006 (link)). A total of 412 particles were extracted and averaged in a box of 44 × 44 × 44 pixels with a pixel size of 6.8 Å. PEET classification was then used to remove misaligned particles (343 final particles). 5-fold symmetry was applied to obtain the final average. The FSC (Fourier Shell Correlation) was generated by using the PEET command calcFSC.

EEL measurements were performed in a STEM (FEI Tecnai F20) with a monochromated 200 keV electron probe of 0.15 eV energy width (full-width-at-half-maximum). EEL spectra were measured with a high resolution Gatan Imaging Filter equipped with a charge-coupled device camera with 2,048 × 2,048 pixel and an energy dispersion of 0.01 eV per channel. The electron beam was scanned over the region of interest and spectra were acquired at each beam position (pixel)27 . The scan step size in the spectrum images varied between 2.9 and 27 nm, the exposure times per EEL spectrum were chosen between 0.05 and 0.1 s. STEM images were acquired with a high-angle annular dark-field detector (Fischione Instruments). The EEL spectra were background subtracted by removing the tail of the zero-loss peak that includes directly transmitted and elastically scattered electrons by using a fitted logarithmic function (‘fitted logarithm tail’ model within Digital Micrograph software (Gatan, USA)).