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 <i>Ma</i><u>g</u>netic <i>I</i>solation and <i>C</i>oncentration (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 <i>Xenopus</i> egg extract. Combining <i>Du</i>plicated <i>S</i>election <i>T</i>o <i>E</i>xclude <i>R</i>ubbish particles (DuSTER), a particle curation method that excludes low signal-to-noise ratio particles, we also resolved the 3D cryo-EM structures of nucleoplasmin NPM2 co-isolated with the linker histone H1.8 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.