Gatan k3 camera

Three to four microliters of the EV solution (1:20 dilution) was added to Lacey carbon grids (300-mesh; Ted Pella, Inc., Redding, CA, USA) that were negatively glow-discharged for 80 s at 30 mA. Excess sample was removed by blotting once for 3 s with Vitrobot filter paper (Ted Pella, Inc, Redding, CA, USA) and then the grid was plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot plunge-freezer (ThermoFisher Scientific, Hillsboro, OR, USA).
The vitrified vesicle samples were imaged using a Talos Arctica 200 kV transmission electron microscope (ThermoFisher Scientific, Hillsboro, OR, USA) equipped with a Gatan K3 camera (Gatan, Inc., Pleasanton, CA, USA) The SerialEM software was used to collect images under low-dose conditions at 36,000 × magnification corresponding to a pixel size of 1.14 Å/pixel. For each image, 50 frames were recorded over 2.5 s exposure time at a dose rate of 35 electrons/pixel/s. The movie frames were aligned using MotionCorr2 (2) under Relion (Zivanov et al. 2018 (link)).

6XHisMBP-PhuN isolated shell fragments were imaged using the 200 kV Glacios microscope. About 34 micrographs were collected manually, six of which were used to pick and extract 9,568 particles. After three rounds of classification, 376 particles remained in the class shown in the Supplementary Fig. 1b inset. The WT PhuN isolated shell fragments were imaged using the 200 kV Talos Arctica (GATAN K3 camera). 10,864 particles were selected from 36 micrographs and processed to a final p2 class with 3,758 particles and p4 class with 1,538 particles (Fig. 1a).