Supplementary Figure 4: Structural comparison emphasizing the local differences and biochemical characterizations of the linker complex used for crystallization.

(a) Structural comparison as in panel c of Supplementary Fig. 3, emphasizing the local differences on H3 and H4. All the differences were seen along the MCM2 HBD binding interface on H3–H4. (b, c) Structural comparison as in panel f of Supplementary Fig. 3, emphasizing a 12^o rotational difference in the α3 helix of H3 between conformations (panel b); and emphasizing the difference in the C-terminal tail of H4 between conformations, which undergoes a disorder to order transition (formation of a βc strand) with ASF1 (panel c). (d) Gel-filtration analysis of the complex with linker (complex 3) and without linker (complex 3’). The complex 3’ was similar to complex 3 except lacking the 12-mer covalent linker. (e) The peak fractions from panel d were analyzed on SDS-PAGE. (f) The tetrasome assembly assay. Lane 1-4, controls. Lane 5, excess H3–H4 tetramer mixed with DNA in physiological salt condition (150 mM NaCl) forming high aggregation. Lane 6, the prepurified complex of MCM2 HBD(43–160)–H3–H4 dimer–ASF1a(1–172) had tetrasome assembly activity. Lane 7, the prepurified complex of MCM2 HBD(61–130)-linker-ASF1b(1–158)–H3–H4 dimer had tetrasome assembly activity on linear Widom 601 DNA, though it shifted the balance among tetrasome and unassigned band (‘*’).