Copper r2 2 200 em grid

After 24 hours of growth, the tube of V. cholerae culture was placed on a bench at room temperature for ~1 min to sediment visible microcolonies. After the pellet was formed the supernatant was gently removed by pipetting and the pellet was resuspended in fresh LB. The cells were incubated for 15 minutes prior to mixing with 10-nm colloidal gold (Sigma-Aldrich, St. Louis, MO) pretreated with bovine serum albumin and subsequently gently applied to freshly glow-discharged Quantifoil copper R2/2 200 EM grid (Quantifoil Micro Tools GmbH, Jena, Germany). The grids were plunge-frozen in a liquid ethane propane mixture⁵⁰ using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Grids were stored in liquid nitrogen prior to imaging.

HD100 cells were grown as described previously (Lambert and Sockett, 2008 (link)) on E. coli S17–1 prey cells in Ca-HEPES buffer at 29°C until most prey cells were cleared from the culture. 10 nm colloidal gold (Sigma-Aldrich, St. Louis, MO) pretreated with bovine serum albumin was added to the cells to serve as fiducial markers during tomogram reconstruction. 3 μl of the resulting sample was pipetted onto a freshly glow-discharged Quantifoil copper R2/2 200 EM grid (Quantifoil Micro Tools GmbH, Jena, Germany) and plunge-frozen in a liquid ethane propane mixture using an FEI Vitrobot mark-III (FEI Company, Hillsboro, OR). The frozen grid was then imaged in an FEI Titan Krios 300 keV field emission transmission electron microscope (FEI Company, Hillsboro, OR) equipped with a Gatan energy filter (Gatan, Pleasanton, CA) and a Gatan K2 Summit direct detector (Gatan, Pleasanton, CA) at the Howard Hughes Medical Institute Janelia Research Campus. Energy- filtered tilt series of images of the cell were collected automatically from −65° to 65° at 1 intervals using the UCSF Tomography data collection software (Zheng et al., 2007 (link)) with total dosage of $100{\text{e}}^{-}/{\text{A}}^2$ , a defocus of −8μm and a pixel size of 4.2Å. The images were aligned and subsequently reconstructed into a tomogram by weighted back-projection method using the IMOD software package (Kremer et al., 1996 (link)).