Extended Data Fig. 1: HTLV-1 Gag/CA sequence and Gag VLP purification.

a) Schematic depiction of the domain architecture of HTLV-1 Gag and the MA₁₂₆CANC truncation protein used for in vitro assembly and structure determination. Residue numbers and black triangles denote protease cleavage sites. Note the absence of non-canonical Gag domains in HTLV-1. b) Sequence of HTLV-1 MA₁₂₆CANC. The positions of α-helices are shown as cyan and yellow bars. Regions for which structural data is lacking are dashed. Triangles denote protease cleavage sites. Please note that due to the used expression and purification system of our 6xHIS-SUMO construct, an ectopic Serine residue remains at the very N-terminus of the purified protein (not shown in this presented sequence). The underlined residues in Helix 9 and 10 denote positively charged residues which could form potential interactions with the NC-RNA. Tryptophan residues (W) are highlighted in green. c) Samples of Optiprep gradient (OP)-purified particles were separated by SDS–polyacrylamide gel electrophoresis. Proteins were visualized by immunoblot using an anti-HTLV-1 p24 antibody. Sucrose pellet (SP), Optiprep bottom (OP), Final Optiprep pellet (F). Positions of molecular mass standards (in kiloDaltons, kDa) are indicated. The molecular weight (MW) of HTLV-1 Gag of 47.5 kDa is annotated. Cell culture production of HTLV-1 Gag VLPs was first established in small scale experiments. The expression was repeated at least 3x with consistent results. The purification blot shown in (c) was performed once to produce the sample in a sufficient quantity to be analysed using cryo-ET.