Cryogenic Electron Microscopy of GBoV1 Complexes

For the GBoV1-Fab complexes, micrographs were collected from frozen grids using a Tecnai G2 F20-TWIN transmission electron microscope (FEI) with a 200 kV voltage under low-dose conditions (20 e⁻/Ų) at a magnification of 82,500× on a 16-megapixel charge coupled device (CCD) camera with pixel size 15 μm, resulting in micrographs with a pixel size of 1.82 Å. This microscope and camera were also used for screening grids for ice quality and particle distribution of GBoV1 prior to high-resolution data collection and also for collecting a low-resolution data set for comparison to the complex structures. For the GBoV1 VLP-alone high-resolution studies, holey carbon grids with vitrified VLPs were used to collect micrograph movie frames on a Titan Krios electron microscope (FEI Co.) operated at 300 kV with a K3 DED using the Leginon application. High-resolution data collection was performed with a total dose of 60 to 67 e⁻/Ų for up to 50 movie frames per micrograph. The movie frames collected on the K3 detector were aligned using MotionCor2 with dose weighting as previously described [37 (link)]. Data sets were collected as part of the NIH project “Southeastern Center for Microscopy of MacroMolecular Machines” (SECM4).