Data for GagΔMAT8I assemblies and GagT8I VLPs were collected at the Electron Bio-Imaging Centre at Diamond Light Source (eBIC-DLS) in the United Kingdom. Tilt-series were acquired on a Gatan K2-Summit detector in super-resolution mode using a Thermo Fisher Scientific KriosG3i microscope operating at 300 kV equipped with a Gatan energy filter (slit width 20 eV; GIF Quantum LS, Gatan). Tilt-series were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 1.35 Å. A dose-symmetric scheme48 was used to collect tilt-series from −60° to 60° or −54° to 54° at a step size of 3° using SerialEM software49. At each tilt, a movie stack consisting of 5–10 frames was recorded with a set dose rate of 3–6 e2/s. Tilt-series were collected at a range of nominal defocus between −1.0 and −4.0 µm and a target total dose of 110–120 e2 was applied over the entire series. For GagΔMAT8I with 10 μM IP6 sample, data were collected at the Thermo Fisher Scientific RnD division in Eindhoven, The Netherlands. Tilt-series were acquired on a Falcon4 detector in EER (electron event representation)50,51 format using a Thermo Fisher Scientific KriosG4 operating at 300 kV equipped with a CFEG and SelectrisX energy filter (slit width 10 eV; Thermo Fisher Scientific). Tilt-series were recorded at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 1.196 Å. A dose-symmetric scheme was used to collect tilt-series from −60° to 60° at a step size of 3° using TEM Tomography software. The CFEG was automatically flashed every 8 h. At each tilt, a movie stack consisting of 217 EER frames was recorded with a dose rate of 4.6 e/px/sec and a total dose of 2.98 e/A2 per tilt. Tilt-series were collected at a nominal defocus range between −1.0 and −4.0 µm and a target total dose of 122 e2 was applied over the entire tilt-series. Further details are given in Table 1.

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