Standard vitrobot filter paper

For cryo-EM, the inactive CASP complex was diluted to 1 μM in a final buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.5% glycerol, and the active CASP complex was used at 1.6 μM in its final storage buffer. Quantifoil R1.2/1.3 300 mesh Cu holey carbon grids (Quantifoil, Germany) were glow-discharged (EMS 100, ElectronMicroscopy Sciences) at 25 mA for 1 min. 3 μl of each sample was applied to glow-discharged grids, blotted for 5 s using Standard Vitrobot Filter Paper (Ted Pella), and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity.

Samples for cryo-TEM were prepared as described above for OMV isolation, with the exception that UC and UF ΔtolA OMV samples were diluted 10-fold in 50 mM HEPES buffer (pH 7.4) due to their higher concentration of OMVs when compared to WT samples. All sets of samples were combined with 10 nm BSA-labeled gold tracer in a 6:1 ratio to assist with automated focusing; 3 μL of this suspension was applied to freshly glow-discharged Quantifoil R 2/2 grids (Quantifoil Micro Tools GmbH, Germany). This suspension was allowed to adhere, and the excess liquid was blotted with standard Vitrobot filter paper (Ted Pella Inc., United States) using a Vitrobot Mark IV (Thermo Fisher Scientific, United States), operating at 5°C and 100% humidity. Grids were then frozen in liquid ethane cooled by liquid nitrogen. Samples were transferred to a Tecnai F20 transmission electron microscope (Thermo Fischer Scientific, United States) using a Gatan 626 DH low-temperature specimen holder (Gatan Inc., United States), and images were recorded using an Eagle 4k CCD camera (Thermo Fischer Scientific, United States). Images were taken in low-dose imaging conditions (10 e/Ų) at both 5,000 and 14,500× magnifications, and vesicle sizes and morphologies were analyzed using ImageJ software version 1.51².