Cryo-EM Analysis of Viral Capsids

Aliquots of the purified CnMV, PBoV1, and RBoV samples were applied to glow discharged, holey carbon grids and the grids vitrified in liquid ethane using a Vitrobot Mark 4 (FEI) at 95% humidity and 4 °C. The grids were screened for the VLP’s particle distribution using a Tecnai G2 F20-TWIN transmission electron microscope (FEI) at 200 kV under low-dose conditions (20 e⁻/Ų) at a magnification of 82,500-fold on a 16-megapixel charge-coupled device (CCD) camera. High-resolution data for the CnMV sample were collected at the Florida State University through the Southeastern Consortium for Microscopy of Macro Molecular Machines (SECM4), using a Titan Krios electron microscope with a DE-64 detector. The microscope was run at 300 kV, with a total dose of 59 e⁻/Ų to collect 52 frames per micrograph. Data for RBoV and PBoV were collected at the Stanford-SLAC Cryo-EM Center (S²C²), using a Titan Krios (FEI) electron microscope operated at 300 kV, equipped with a Falcon 4 direct electron detector (Thermo Fisher). A total of 50 movie frames were collected per micrograph at a total electron dose of ~50 e⁻/Ų. The movie frames were aligned using MotionCor2, as described previously [31 (link)].