From each of the three groups of LANA-DBM-nucleosome binding poses, two structures were selected (Complexes 1-6 in Figure , the detailed selection process of these poses is described in Supporting Information), and MD simulations in explicit water box were performed. During the 150-ns-long simulations, Complexes 3-6 involving DNA groove-DBM interactions maintained their initial structures (Figure ). On the other hand, one of the poses involving DBM bound to histone surface (Complex 1), changed the sitting position of DBM to DNA major groove at SHL -3.5 (Figure ). The other pose (Complex 2) maintained DBM binding to the histone surface, but its binding free energy calculated by the generalized Born surface area (MM-GBSA) method 20 was significantly lower than those of other trajectories involving DBM-DNA groove interactions (Table ). These observations suggest that DBM binding on the histone surface is not stable compared to DBM binding to DNA grooves in water. Consequently, we postulate DNA major groove at SHL -4.5 and minor groove at SHL -5 to be probable DBM binding sites (Figure ). Of note, a previous report showed that these DNA grooves have fewer contacts with histone tails, and thus, are highly accessible by other molecules. Among the structures with DBM -DNA groove interactions, Complex 4 involving DBM bound to DNA major groove at SHL -4.5, furnished an apparently lower binding free energy than the others (Table ), suggesting a high contribution of this conformation (Figure ). DNA major groove at SHL -3.5 and its surrounding grooves are also possible DBM binding sites, as shown in the structure of Complex 1 after 150ns simulation. However, given that its binding free energy is relatively high (Table ) and these grooves are supposed to be highly hindered by interaction with H2A/H2B tails, the contribution of DBM binding to these grooves might be small.