Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination

Abstract

Summary: 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).

Keywords

• Molecular Biology
• Antibody
• Protein Biochemistry
• Protein expression and purification
• Structural Biology
• Cryo-EM