Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination
Bing‐Rui Zhou, Yawen Bai
Abstract
The chromatosome, a nucleosome bound to a histone H1, is the structural unit of metazoan chromatin. Determination of the high-resolution structure of the chromatosome is challenging due to the dynamic nature of H1 binding. Here, we present a protocol for purifying an optimized single-chain antibody variable fragment (scFv) that can be used to stabilize the chromatosome for single-particle cryo-EM studies. This protocol facilitates high-resolution cryo-EM structure determination of nucleosomes with a natural DNA sequence, chromatosomes, and other protein nucleosome complexes. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2021).
Topics & Concepts
NucleosomeCryo-electron microscopyHistoneChromatinDNABiophysicsSequence (biology)Resolution (logic)ChemistryParticle (ecology)Computational biologyBiologyComputer scienceBiochemistryArtificial intelligenceEcologyAdvanced Electron Microscopy Techniques and ApplicationsRNA and protein synthesis mechanismsEnzyme Structure and Function