Molecular Architecture of the H2AX-Containing Nucleosome from Arabidopsis

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 remains poorly understood. Here, we determine cryogenic electron microscopy structures of an Arabidopsis thaliana (At) 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 the 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.