Cryo-electron Tomography of TAILS

Microscopy was performed as described previously^{46 (link),54} . In brief, images (electron dose of ~25 e⁻/Ų; −4 to −6 µm defocus) were acquired at 27500 x on a Tecnai F30 electron microscope (FEI Company Ltd) operated at 300 kV. The detector was a GATAN UltraCam, lens-coupled, 4 K CCD camera (binned by 2) attached to a Tridiem Gatan Image Filter (GIF: operated at zero-loss mode with an energy window of 20 eV; Gatan Inc., Pleasanton, CA, USA). For tomography, tilt-series were acquired every 1.5 degree (±60 degrees) using serialEM software^{55 (link)}. The total electron dose was kept between 80–120 e⁻/Ų. Cryo-ETs were calculated using eTomo and CTF correction was applied to all datasets. All tomograms were acquired on cells from a single donor and cryo-electron micrographs were acquired from the other two donors to confirm the presence of TAILS.