Molecular Architecture of the Arabidopsis H2AX-Containing Nucleosome

Nucleosomes integrate diverse histone variants to organize chromatin and coordinate genome maintenance. In plants, the structural interplay between replication-associated histones and DNA damage -responsive histone variants remain poorly understood. Here, we determine cryogenic electron microscopy structures of an Arabidopsis thaliana nucleosome containing the replication-associated histones H2B.6 and H3.1 together with the DNA damage-responsive variant H2AXa.The mononucleosome structure, resolved at 2.73 Å preserves the canonical nucleosome architecture. Unexpectedly, the reconstituted mononucleosomes formed defined higher-order assemblies in the absence of linker DNA or chemical crosslinking. Two distinct nucleosome-nucleosome arrangements, parallel and right offset, were identified. The right offset closely resembles the human counterpart, whereas the parallel arrangement exhibits slight displacement of H2B structural elements at the inter-nucleosomal interface. Together these structures reveal how a distinct histone composition preserves canonical nucleosome architecture while supporting multiple defined modes of nucleosome packing, providing structural insights into conserved and divergent features of H2AX containing chromatin organization across plants and animals.