Particle production and purification

The Gag_ΔMAT8I mutant was constructed from pET2 PRR400 by site-direct mutagenesis. Protein was expressed in E. coli, Rosetta 2 (DE3), cultured in Luria-Bertani medium, and induced with 0.5 mM isopropylthiogalactoside at 23 °C for 16 h. Gag_ΔMAT8I assemblies were formed inside of bacterial cells and purified directly from cell lysate. The cell pellets were collected and resuspended in lysis buffer (25 mM Tris, pH 7.5, 0.5 M NaCl) and broken with a microfluidizer. Subsequently, the lysate was centrifuged at 5000 × g for 10 min to remove cell debris. The supernatant was collected and subjected to sucrose gradient centrifugation (30–70%) with a 15% sucrose cushion on the top. Gradient was spun at 210,000 × g for 18 h. Particles were collected from the pellet and resuspended in lysis buffer with and without 10 μM IP6 and used for CryoEM.

The GagT8I mutant VLPs were produced from HEK293T cells (ATCC CRL-3216) transfected with a codon-optimized full-length Gag expression plasmid (pCMV-Gag-opt)⁴⁷. The culture supernatants were harvested approximately 40 h after transfection, filtered through a 0.45-micron pore-sized filter, and pelleted through a cushion of 20% sucrose (wt/vol) in STE buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA). The VLPs were gently resuspended in STE buffer and frozen at −80 °C.