Supplementary Figure 1: Biochemical characterization of different MCM2 HBD–H3–H4 complexes and structural comparisons.

(a) His-pulldown of MCM2 HBD with recombinant histones. (b) The MCM2 HBD(61–130)–H3.3(Δ56) –H4 tetramer (complex 1) and MCM2 HBD(43–160)–H3.3(Δ56) –H4 tetramer (complex 2) were analyzed by gel-filtration assay. (c) SDS-PAGE analysis of the peak fractions from panel b. (d) The MCM2 HBD(43–160)–H3.2–H4 tetramer (complex 6), MCM2 HBD(43–160)–H3.2–H4 dimer–ASF1a(1-172) (complex 5) and H3.2–H4 tetramer were analyzed as in panel b. (e) SDS-PAGE analysis of the peak fractions from panel d. (f) SEC-MALS assay. The apparent Mw determined by SEC-MALS of MCM2 HBD–H3–H4 tetramer complex is 93.5 kDa (errors 3%, with the expected Mw about 80.0 kDa). The apparent Mw of MCM2 HBD–H3–H4 dimer–ASF1 complex is 64.3 kDa (errors 4%, with the expected Mw about 60.0 kDa). (g) A stereo view following superposition of the two halves of the MCM2 HBD–H3–H4 tetramer complex. One half was in color and the other half was in silver. (h) The reported structure (Richet, N. et al., Nucleic Acids Res 43, 1905-1917, 2015) observed a 1:1:1 MCM2 HBD–H3.2–H4 dimer complex in the asymmetric unit (AU) and a ‘2:2:2’ complex could be generated through crystallographic symmetric operation, while our structure directly observed a 2:2:2 MCM2 HBD–H3.3–H4 tetramer complex in the AU. Panel h showing the superposition of the generated ‘2:2:2’ structure (in silver) with our observed 2:2:2 structure (in color) with an rmsd of 1.3 Å. The major differences in conformations are pointed out with arrows. (i) A stereo view following superposition of the H3–H4 tetramers from the structures of MCM2 HBD–H3-H4 tetramer complex (in color) and nucleosome (in silver). The H2A docking domain from the nucleosome was also shown.