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. 2020 Aug 26;11:4263. doi: 10.1038/s41467-020-18067-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Domain architecture of DmORC subunits and Cdc6. Dashed lines demarcate regions that are flexible and structurally not resolved. Gray regions in Orc1 and Cdc6 were removed to improve sample behavior for cryo-EM. The color scheme is maintained throughout the figures unless noted otherwise. b Purification of DmORC·DNA·Cdc6 for cryo-EM. Chromatograms are shown for the ternary complex (solid lines) and for isolated DNA (dashed lines). Peak fractions retain all DmORC subunits and DmCdc6 when analyzed by SDS-PAGE (inset). The asterisk corresponds to a degradation product of Orc2 and/or Orc3. c Side view of the unsharpened cryo-EM map with AAA+ and WH domains colored differentially, highlighting the two-tiered organization of the complex. d Near-planar arrangement of the AAA+ and WH modules with respect to the main DNA axis (gray line) in the ORC·Cdc6 ring. The centers of mass of the AAA+ base and WH domains are depicted as spheres. e AAA+ and WH view of the cryo-EM density (unsharpened) with ORC and Cdc6 subunits colored differentially. The gap between Orc3 and Orc2 in the WH layer is indicated. f, g Conformational rearrangements in DmORC during the transition from the autoinhibited to the activated state. In f the positions of the Orc1 AAA+ and Orc2 WH domains in the autoinhibited apo-DmORC state (shown as cartoon; PDB 4xgc¹⁶) and the ORC·DNA·Cdc6 complex (ODC; depicted as molecular surface) are compared after structural alignment of both assemblies. Arrows indicate the domain movements during activation. Side views are shown, and other subunits/domains are displayed as light gray cartoon. g ATP binding to Orc1 reorients the Orc1 AAA+-lid and closes the Orc1/4 interface. The Orc1 AAA+-base subdomain of Orc1 in apo-DmORC (PDB 4xgc¹⁶) was superposed on the same region in the ternary complex. BAH, bromo-adjacent homology domain; IDR, intrinsically disordered region; BP, basic patch; WH, winged-helix; NTD, N-terminal domain; CTD, C-terminal domain; TFIIB, transcription factor IIB-like domain. Source data are provided as a Source data file.