Tecnai g2 f20 twin transmission electron microscope

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)].

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).

The 5′-biotinylated 80 nt ssDNA substrate for gold labeling was synthesized and purified (IDT). The streptavidin-colloidal nanogold (10 nm, Sigma-Aldrich) and biotinylated 80 nt ssDNA were allowed to react for 1 h at room temperature, which generated a 5′-end-labeled with 10 nm nanogold particles coupled to streptavidin. The hRAD51–ADP double-filaments were prepared by incubation of hRAD51 (400 nM) with the 5′-nanogold-labeled ssDNA (2.5 nM) in the presence of 1 mM ADP at room temperature for 20 min. The protein-DNA complexes were applied to glow-discharged carbon-coated grids. The grids were negatively stained with 1% uranyl acetate for 1 min, and images were obtained from the FEI Tecnai G2 F20 TWIN transmission electron microscope.