Structural Analysis of HIV Env Trimer Complexes

SEC-purified BG505 SOSIP.664 gp140 trimers, either alone or as Fab complexes (with b6, F240, 14e, 19b, PGV04, sCD4/17b), were analyzed by negative stain EM. A 3 µL aliquot containing ∼0.03 mg/mL of the trimer or Fab-trimer complex was applied for 5 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, then negatively stained with Uranyl formate or Nano-W (Nanoprobes) for 30 s. Data were collected using a FEI Tecnai F20 or T12 electron microscope operating at 120 keV, with an electron dose of ∼55 e⁻/Ų 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 Gatan US4000 CCD or Tietz TemCam-F416 CMOS camera using a nominal defocus range of 900 to 1300 nm.