MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples

Cryo-EM single-particle analyses typically require target macromolecule concentration at 0.05∼5.0 mg/ml, which is often difficult to achieve. Here, we devise Magnetic Isolation and Concentration (MagIC)-cryo-EM, a technique enabling direct structural analysis of targets captured on magnetic beads, thereby reducing the targets’ concentration requirement to < 0.0005 mg/ml. Adapting MagIC-cryo-EM to a Chromatin Immunoprecipitation protocol, we characterized structural variations of the linker histone H1.8-associated nucleosomes that were isolated from interphase and metaphase chromosomes in Xenopus egg extract. Combining<underline>Du</underline>plicated<underline>S</underline>election<underline>T</underline>o<underline>E</underline>xclude<underline>R</underline>ubbish particles (DuSTER), a particle curation method that removes low signal-to-noise ratio particles, we also resolved the 3D cryo-EM structures of H1.8-bound nucleoplasmin NPM2 isolated from interphase chromosomes and revealed distinct open and closed structural variants. Our study demonstrates the utility of MagIC-cryo-EM for structural analysis of scarce macromolecules in heterogeneous samples and provides structural insights into the cell cycle-regulation of H1.8 association to nucleosomes.