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To further improve the resolution by overcoming the heterogeneity of the sample and by correcting the defocus gradient from various parts of the VLP, we divided the icosahedral reconstructions into blocks of 2-fold (2f), 3-fold (3f), and 5-fold (5f) symmetry using a block-based algorithm 42 and separately calculated their reconstructions with subparticles re-extracted, followed by localized refinement

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Freeze individual doses (controversial) Some users pre-load syringes and freeze them individually