A homemade manual plunger was used for cryoEM grid preparation. Quantifoil R2/2 copper grids were freshly glow discharged at 30 mA for 30 s. For GagΔMAT8I assemblies, 2 µl sample was applied to the carbon-side of the grid, followed by 1 μl 10 nm fiducial gold bead solution, and blotted from the backside since samples are very fragile and sensitive to blotting paper. 1 μl sample buffer was then added on the grid backside to facilitate the filter paper blotting from the backside. For GagT8I VLPs, 1 μl 10 nm fiducial gold beads were applied to the grid first, followed by 1~2 μl sample, and blotted from the frontside. The grid after blotting was then quickly immersed into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until data collection.

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