Cryo-EM analysis on magnetic beads for scarce macromolecules in heterogeneous samples.
Overview
abstract
Here, we introduce Magnetic Isolation and Concentration (MagIC)-cryo-EM, a technique enabling direct structural analysis of targets captured on nanomagnetic beads, thereby reducing the targets' concentration requirement to < 0.0005 mg/ml, which is 100 to 1000-fold lower than conventional cryo-EM methods. Using MagIC-cryo-EM, we characterized structural variations of oocyte-specific linker histone H1.8 associated nucleosomes isolated from interphase and metaphase chromosomes in Xenopus egg extract. In combination with Duplicated Selection To Exclude Rubbish 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, revealing distinct open and closed structural variants. Our approaches offer structural insights into how the chaperone NPM2 interacts with its targets and the cell cycle-dependent regulation of H1.8 association to nucleosomes.
