UFO-BG trimers, UFO-C trimers, and gp140 trimer-presenting nanoparticles were analyzed by negative-stain EM. A 3-μl aliquot containing trimers or nanoparticles (~0.01 mg/ml) was applied for 15 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s and then negatively stained with 2% (w/v) uranyl formate for 30 s. Data were collected using a FEI Tecnai Spirit electron microscope operating at 120 kV, with an electron dose of ~25 e Å−2 and a magnification of ×52,000 that resulted in a pixel size of 2.05 Å at the specimen plane. Images were acquired with a Tietz 4k × 4k TemCam-F416 complementary metal-oxide semiconductor (CMOS) camera using a nominal defocus of 1500 nm and the Leginon package (109). UFO-BG trimer particles were selected automatically from the raw micrographs using DoG Picker (110), while trimer-presenting nanoparticles were selected manually using the Appion Manual Picker (111). Both were put into particle stack using the Appion software package (111). Reference-free 2D class averages were calculated using particles binned by two via iterative multivariate statistical analysis/multireference alignment and sorted into classes (112). To analyze the quality of the trimers (native-like and non-native), the reference-free 2D class averages were examined by eye, as previously described (26).

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