Cryo-EM Sample Preparation for Bacterial Cells

Plunge freezing was performed using an FEI Vitrobot (Thermo Fisher)^{53 (link)}. For cryoET sample preparation of bacterial cells, 10 nm colloidal gold fiducial markers (Sigma-Aldrich) were added to each sample at a ratio of 1:5 (v/v) to allow tilt image alignments. For Vitrobot setup, a filter paper (Whatman, 47 mm diameter) and a Teflon sheet were installed for single-sided blotting in a pre-cooled chamber (4 °C) with 100% humidity. EM grids (R2/2, Cu 200 mesh; Quantifoil Micro Tools) were glow-discharged for 45 s at 25 mA by PELCO easiGlow discharger. Sample aliquots (4 μl) were applied to each grid, incubated for 15 s and blotted for 6.5 s, followed by immediate plunge freezing in an ethane:propane mixture (37% v/v ethane:63% v/v propane)^{54 (link)}. Grids were stored in liquid nitrogen. Before loading of the samples into the cryo-electron microscope, the grids were clipped. For sample preparation, all bacterial samples were pelleted, and OD₆₀₀ was adjusted to 2–2.5 before blotting. If required, L. monocytogenes or E. faecalis cells were exposed to 1,024 nM purified Ply006 or Ply007, respectively, followed by plunge freezing at the desired timepoints. For imaging of phage adsorption, bacterial cultures were adjusted to an OD₆₀₀ of 0.1. Samples (95 µl) were then mixed with 5 µl of purified phage lysate (10¹¹ p.f.u. ml⁻¹), followed by 5 min incubation at room temperature.