Extended Data Fig. 8: ORC in MO is perfectly positioned for loading the second MCM ring in the correct orientation for the formation of the double hexamer.

a, Negative-stain and cryo-EM 2D classes, and 3D structures of OCCM (top) and MO (bottom), with the loading intermediates aligned via their respective ORC complexes. b, Three-dimensional model, based on a, of the proposed mechanism for recruitment of the second MCM. OCCM is shown superposed to the ORC of MO. This superposition places a second MCM–Cdt1 such that its Mcm2–Mcm5 gate is oriented for threading duplex DNA into the MCM channel. c, Negative-stain 2D class showing a post-MO loading intermediate, captured by supplementing MO complexes with MCM–Cdt1 before imaging. This class appears to be a second MCM recruitment complex, containing MO and an additional MCM–Cdt1. d, A cryo-EM 2D class average of the post-MO complex (top) shows bent duplex DNA aligned to the Mcm2–Mcm5 DNA gate of the second MCM–Cdt1, captured before DNA threading. This is the same configuration that was previously identified for the OC–MC complex (middle). Alignment of the OC–MC and MO 2D classes by their respective ORC complexes matches the observed configuration of the second MCM recruitment complex, MOC–MC. e, Three-dimensional model of MOC–MC, based on the MCM-Cdt1 structure¹⁰, the MO structure (this study) and 2D class averages shown in c and d. f, Cdc6 is required for the loading of the second MCM helicase. Following immunodepletion of Flag-tagged Cdc6, MO is unable to load a second MCM; this results in a failure to form salt-stable double hexamers on DNA in the absence of additional Cdc6. For gel source data, see Supplementary Fig. 1.