Supplementary Figure 1 Purification, SDS-PAGE and cryo-em characterization of the MCM hexamer and Cdt1 MCM heptamer samples. (a-b) SDS-PAGE analysis of the hexamer and heptamer samples. The eluted hexamer (a) and heptamer (b) complexes were subjected to 20 40% glycerol gradient sedimentation centrifugation. Collected fractions were analyzed by SDS-PAGE and visualized by coomassie blue staining. Molecular size markers used are: ALP 140 kda and thyroglobulin 669 kda. Fractions 7-9 (a) and 8-10 (b) were pooled and concentrated for cryo-em analysis. (c) The sedimentation profile of Mcm5 and Cdt1 from the hexamer (a) and heptamer (b) samples were plotted using quantified signals by Image J. (d) A representative raw micrograph of Cdt1 Mcm2-7 complexes in vitreous ice. (e-g) Representative 2D class averages of cryo-em particles from the datasets of heptamer+amppnp (e), heptamer+adp (f), and hexamer+adp+np40 (g). See also Table 1.
Supplementary Figure 2 Flowchart for image processing of particles from the heptamer + AMPPNP data set and Fourier shell correlation curves of the five structures. (a) A flowchart of the steps in image processing. (b-f), Fourier Shell Correlation curves of the heptamer AMPPNP (b), heptamer ADP (c), hexamer AMPPNP (d), hexamer ADP (e) and (hexamer ADP)* (f) structures. Note that (hexamer ADP)* is the hexamer structure from the dataset of heptamer + ADP. See also Table 1.
Supplementary Figure 3 Overall structures of the heptamer and hexamer. (a-c) Top panels, The cryo-em maps of the indicated complexes are shown in surface representation, with individual components color coded. Bottom panel, the maps are shown in transparent surface representation, with models superimposed. Both the CTD top-view
and consecutively rotated side-views are shown. a, hexamer AMPPNP. b, heptamer ADP c, hexamer ADP.
Supplementary Figure 4 Arrangements of oligonucleic-acid-binding subdomains (OBs) in the heptamer and double hexamer. (a-b) Arrangements of OB subdomains from the heptamer (a), and the double hexamer (b). Models are shown in ribbon representation. Individual OB subdomains are color-coded. (c-d) Superimposition of OB subdomains from heptamer (color-coded) and double hexamer (grey), shown in top (c) and side (d) views. The alignment is done using the OB of Mcm7 as reference.
Supplementary Figure 5 Constellation of Cdt1-binding sites in the heptamer and double hexamer. (a) The binding sites of Cdt1 on the NTD of Mcm2 (orange), CTD-A of Mcm6 (blue) and NTD of Mcm4 (magenta) as well as the two interacting N-C-linkers of Mcm6 (sky blue) and Mcm4 (red) in the heptamer are highlighted in different colors. (b) The MCM single hexamer from the double hexamer is aligned to the heptamer and shown in a similar orientation as in (a), with Cdt1 binding sites mapped. The NTD of Mcm6 (tan) is used as reference for the alignment.
Supplementary Figure 6 Structural comparison of the heptamer and double hexamer. (a-f) Conformational differences of each NTDs from the heptamer and the double hexamer. NTD-A was used as reference for the alignment. NTDs of the heptamer are color coded and the corresponding NTDs in the DH are in grey. Dramatic changes are observed for the ZF motifs of Mcm2 (20 Å) and Mcm3 (10 Å). The distance changes of the other motifs of NTDs (ZF and OB) are measured by r.m.s.d. (root-mean-square deviation). r.m.s.d. values: M2 (7.5 Å), M6 (1.9 Å), M4 (2.0 Å), M7 (1.6 Å), M3 (3.5 Å), M5 (2.2 Å). (g-k) Conformational differences of the CTD-A dimers in the heptamer (color-coded for subunits) and in the double hexamer (grey). One CTD-A of each neighboring pairs (the left one) was used as the reference for alignment. Calculated r.m.s.d values of the other CTD-As are: M6 (4.3 Å), M4 (6.0 Å), M7 (6.8 Å), M3 (6.1 Å), M5 (5.0 Å).
Supplementary Figure 7 Cdc45 and GINS are structurally incompatible for interactions with MCM subunits in the heptamer and open- and closed-ring conformations of different MCM complexes. (a) Structure of the CMG complex in surface representation. The map of the CMG was converted from a previous atomic model (PDB 3JC5). Mcm5, Mcm2, GINS, Cdc45 are colored yellow, orange, sky blue and purple, respectively. (b) Structure of the Cdt1 Mcm2-7 heptamer, displayed in a comparable orientation as in (a). (c-d) Superimposition of Cdc45 and GINS from the CMG structure onto the map of the heptamer, showing in top (c) and side (d) views. Mcm2-NTD was used as reference for the alignment. As shown, aligned
Cdc45/GINS exhibits structural conflict with the CTD-A of Mcm5 in the heptamer, indicating that the interactions of Cdc45/GINS with the NTDs of Mcm5 and Mcm2 observed in the CMG complex could not be simultaneously satisfied in the heptamer. (e) CTD top views of the cryo-em maps (grey) of the yeast MCM hexamer, Cdt1 MCM heptamer, and double hexamer (EMD-6338). The double hexamer map is low-pass filter to 10 Å. (f) CTD top views of the two conformers of the yeast CMG complex (EMD-6536 and EMD-6535). (g) CTD top views of the two conformers of the Drosophila CMG complex (EMD-3321 and EMD-3320).