Glow discharged c flat holey carbon grid

VLP samples were prepared for cryo-TEM analysis as previously described [26 (link)]. Briefly, concentrated VLP samples were incubated on a glow-discharged c-flat holey carbon grid (Ted Pella, Redding, CA, USA), and blotted with filter paper to remove excess sample. Grids were quickly frozen in liquid ethane [38 (link)] with a FEI Vitrobot MarkIII system. A FEI TF30 field emission gun transmission electron microscope at liquid nitrogen temperature (FEI Company, Hillsboro, OR, USA) was used to analyze frozen samples. Imaging of samples was done at a nominal magnification of 59 k at low-dose (~30 electrons/Ų) and 1 to 5 µm underfocus conditions using a Gatan 4 k by 4 k charged coupled device (CCD) camera (Gatan Inc., Pleasanton, CA, USA). The fluorescence image and corresponding TEM picture were recorded using an FEI iCorr installed on a FEI Tecnai Spirit TEM.

Virus and VLP samples were prepared for cryo-TEM as previously described [21 (link)]. Briefly, 3 µL concentrated virus or VLP sample was applied to a glow-discharged c-flat holey carbon grid (Ted Pella, Redding, CA, USA) and then blotted with filter paper to remove the sample excess. The grid was then plunged frozen into liquid ethane [25 (link)] with a FEI MarkIII Vitrobot system (FEI Company, Hillsboro, OR, USA). The frozen grids were then transferred to a FEI TF30 field emission gun transmission electron microscope at liquid nitrogen temperature (FEI Company). Images were then recorded at a nominal magnification of 39,000_x and 59,000_x at low-dose (~30 electrons/Ų) and 1 to 5 µm under focus conditions using a Gatan 4 k by 4 k CCD camera (Gatan Inc., Pleasanton, CA, USA).