Leica em gp system

Overnight cultures of E.coli strains were diluted in LB 1:100 and grown at 37°C. At OD₆₀₀=0.2, 150 μM IPTG was added to the liquid culture to induce CRISPRi knockdown. Bacteria were grown for another 90 min and then flash-frozen in liquid nitrogen. Cell cultures were mixed with 10 nm protein A gold at 20:1 ratio (Utrecht), then aliquots of 3 μL mixtures were applied to glow-discharged R2/2, 200 mesh copper Quantifoil grids (Quantifoil Micro Tools). The sample was blotted for 3 s at 20°C and at 80% humidity. The grids were plunge-frozen in liquid ethane using Leica EM GP system (Leica Microsystems) and stored in liquid nitrogen. Cryo-ET was performed on a Talos electron microscope equipped with a Ceta CCD camera (ThermoFisher). Images were taken at magnification 22,000x corresponding to a pixel size of 6.7 Å. Tilt series were collected using SerialEM^{79 (link)} with a continuous tilt scheme (−48° to 48°, every 3° increment). The defocus was set to −6 to −8 μm and the cumulative exposure per tilt series was 150 e⁻/A². Tomograms were reconstructed with the IMOD software package^{80 (link)}.

B. subtilis cells were grown to an O.D. of 0.5 and 1 ml of B. subtilis cells were mixed with 5 μl S. coelicolor spores glycerol stock and incubated at ambient temperatures for 5 min. Cells were concentrated by centrifugation and 3 μl aliquots of the cell suspension are applied to glow-discharged R2/2 200 mesh copper Quantifoil grids (Quantifoil Micro Tools), the sample was pre-blotted for 30 s, and then blotted for 2 s. Grids were pre-blotted and blotted at 20 °C and at 95% humidity. The grids were plunge frozen in liquid ethane using an automated Leica EM GP system (Leica Microsystems) and stored in liquid nitrogen. The grids were imaged on a 120 kV Talos L120C cryo-electron microscope (Thermo Fisher Scientific) at the Netherlands Center for Electron Nanoscopy.