E2-ubiquitin (Ub) conjugates are useful chemical biology tools for studying the E3 mechanisms, but so far, the reported systems have involved either no substrates or only short peptide substrates. Here, we describe a practical chemical synthesis of E2-Ub-nucleosome conjugates that can trap the transient intermediates of E3- mediated Ub transfer from E2 to nucleosomes to form stable E3/ E2/nucleosome complexes, whose characterization and structure determination revealed how distinct E3-E2 modules establish a specific architecture to orient the Ub toward the target lysine. Our work provides the first example to support the use of a chemical trapping strategy to study the E3-mediated Ub-transferring mechanisms on full-length and folded proteins and overcomes the possible limitations of the linear E3/E2 fusion strategy—that head-to-tail fusion may preclude the optimal interaction of E3 and/or E2 with the substrate and that delineation of where E2 is head-to-tail fused is difficult when E3 is composed of multiple subunits.