HIV Env proteins were visualized by negative-stain electron microscopy (EM) using 3–4 µL aliquots at concentrations of 0.1–0.2 mg/mL. Samples were applied for 10 s onto a mica carbon film and transferred to 400-mesh Cu grid that had been glow discharged at 20 mA for 30 s and then negatively stained with 2% (wt/vol) Sodium silicotungstate (SST) for 30 s. Previous to data collection, the grids were screened to assess stain quality and particle distribution. Data were collected on an FEI Tecnai T12 LaB6-EM operating at 120 kV accelerating voltage with a pixel size of 2.8 Å on the specimen plane using a Gatan Orius 1000 CCD Camera. On average, 30–40 micrographs were collected per sample. Classification of closed and open trimers was performed as described [16 (link)]. Briefly, two-dimensional (2D) class averaging was performed with the software Relion [37 (link)], using 35,829 particles for the analysis of ConCv2 KIKO; 56,407 particles for ConCv4 KIKO; and 55,248 particles for ConCv5 KIKO. The 2-D classes were then segregated into three structural groups, closed or open-native particles and non-native particles.
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