Cryo-EM Structural Analysis of SIVmac239 Env Trimer

SIV_mac239 Env trimers were affinity purified with PGT145 and analyzed by cryo-electron microscopy. Three microliters of ∼6-mg/ml Env trimer was diluted 3:1 into 1.8 mm n-dodecyl-β-d-maltoside and plunge frozen on 1.2/1.3 C-Flat holey carbon grids (Protochips) with an FEI Vitrobot Mark IV (Thermo Fisher). Data were collected on an FEI Titan Krios instrument (Thermo Fisher) operating at 300 keV at a defocus range of –0.6 to −2.6 μm and controlled using Leginon (34 (link)). Movie micrographs were captured on a K2 Summit direct electron detector (Gatan) using an electron dose rate of ∼4.65 e⁻/pixel/s, a 250 ms⁻¹ frame rate, and a total exposure time of 11.5 s, for a total dose of ∼50.4 e⁻/Ų. Frames were aligned and dose weighted with MotionCor2 (35 (link)), and the contrast-transfer functions were estimated with Gctf (36 (link)). The final magnified pixel size was 1.03 Å. Particles were picked and extracted with Relion2 automated particle picking using a Gaussian disc as a template (37 (link)). 2-D classification was performed using CryoSparc2 (38 (link)). The final set of 2-D classes shown in Fig. S1 in the supplemental material is composed of ∼62,000 particles.