Fig. S7.

Models of dimer dissociation reported by two different FRET pairs. (A and B) NCP structure (1AOI) highlighting the distances between the FRET pairs: H3-78W donor to H2B-109CA acceptor (H3–H2B NCP) (A) and H4-80W donor to H2A-108CA acceptor (H4–H2A NCP) (B). Due to the symmetry of the structures in both constructs, distances are mirrored between the FRET pairs on both sides. Note: The Förster radius R₀ ∼20 Å. The distances for the D–A pair (on the same NCP face) and D–A′ pair (across the NCP) are similar for the H4–H2A NCP. Although equal changes in FRET signals are expected for the D–A and D–A′ interactions, the dissociation of a single H2A–H2B dimer should still be reflected by the loss of ∼50% of the FRET signal due to the symmetry of the construct. Below each model in A and B are the measured FRET amplitudes (as reported in Table 1). (C–F) Models of possible histone configurations to explain the amplitude patterns observed for H3–H2B NCP (C and D) and H4–H2A NCP (E and F). For each FRET pair, the expected amplitude changes are shown for the scenarios that the open intermediate is formed asymmetrically (C and E) and symmetrically (D and F). The measured amplitude patterns differ between H3–H2B NCP (33% → 47%) and H4–H2A NCP (47% → 30%). Release of the first H2A–H2B dimer may slightly destabilize the remaining H2A–H2B dimer (depicted as a slight separation) through the loss of some minor contacts between the two H2A–H2B dimers. The H4–H2A NCP construct may be more sensitive to this destabilization due to the positions of the FRET pair. Our data are most consistent with the sequential formation as follows: canonical NCP → asymmetric open intermediate → hexasome → tetrasome.