Electron microscopy was performed by Tokai-EMA based on rapid freezing and freeze-fixation methods. Cells were sandwiched with copper disks and quickly frozen in liquid propane at −175°C. The frozen samples were substituted with 1% tannic acid in ethanol and 2% distilled water at −80°C for 48 h and then and kept at −20°C for 4 h and at 4°C for 1 h. These samples were dehydrated in anhydrous ethanol for 30 min three times and infiltrated with a 50:50 mixture of ethanol and LR white resin (London Resin) at 4°C for 1 h. After infiltration, the samples were washed with LR white three times by incubation at 4°C for 30 min for each wash process and transferred to fresh LR white, followed by polymerization at 50°C overnight. The polymerized resins were ultrathin sectioned, and the sections were placed on nickel grids and immunostained with anti-Nsp1 antibodies and secondary antibodies conjugated to 10-nm gold particles as reported previously (Shima et al., 2019 (link)). Electron microscopy was performed using a transmission electron microscope (JEM-1400Plus; JEOL), and digital images (3,296 × 2,472 pixels) were obtained using a charge-coupled device camera (EM-14830RUBY2; JEOL).