(A - C) Radiolabeled Orc1/2/3/4/5 (A), or deletion constructs of Orc2 (B, C) were expressed using an in vitro transcription/translation system and pulled down by GST (G) or GST-Orc6 (6). Input (5%) and bound materials (30%) were visualized. V, expression vector; FL, full length. (D) Comparison of reference-free class averages of EM images of ORC and Orc1-5 subcomplex. Image adapted from (Chen et al.). (E) A complete assignment of all six subunits of ORC.

(A) A raw image of ORC mixed with 66-bp ARS1 dsDNA at 1:1.2 molar ratio. Several individual particles are marked in white circles. (B) Selected 2D reprojections (left) and corresponding reference-based class averages (right) of the ORC-dsDNA complex. (C) Cryo-EM 3D map of ORC alone in top (upper) and front side (lower) views. (D) Cryo-EM 3D map of ORC-dsDNA shown in top (upper) and front side (lower) views. Individual subunits are labeled. See also for 3D reconstruction details.

(A) Negatively stained EM image averages of ORC-DNA and (B) Cdc6-ORC-DNA. Gain of density in the presence of Cdc6 is marked by filled arrows in (B). Orc1 re-arrangement upon Cdc6 binding is indicated by white arrows. (C) Comparison of cryo-EM 3D map of ORC-DNA with that of ORC-DNA-Cdc6 shown in (D). The precise boundary between Orc2 and Orc3 cannot be determined based on the current data. Orc1 density is painted in light blue, and Orc1 NTD painted in cyan. Cdc6 density in (D) is painted in magenta. Rearrangement of Orc1 density upon Cdc6 binding is illustrated by a pair of blue arrows. The protruding density in ORC-Cdc6-DNA (D) is painted in red and tentatively assigned to be a part of Orc6. See also for 3D reconstruction details.

Radiolabeled Orc1 (FL) or its deletion constructs were expressed in vitro and pulled down by GST-Orc6 (Panel A and 6 in panel B) or GST (G in panel B). Input (5%) and bound materials (30%) were visualized.

(A) Crystal structure of archaeal Orc1/Cdc6 in the absence of DNA in cartoon view showing the three linearly arranged domains (). (B) Archaeal Orc1 in DNA binding conformation in cartoon view showing the highly curved C-shaped arrangement of the three domains. DNA molecule is not shown for clarity. Two pink ellipses mark the approximately DNA binding regions in the N-terminal α/β-folded ATPase domain and the C-terminal WHD (). (C) Segmentation of the 3D map. Each subunit is shown in a different color. Segger () as implemented in Chimera () was used to segment the density. Cdc6 at the side, Orc1, Orc4 at the top, and Orc5 in the middle are more separated, their boundary definition is objective. Orc2, Orc3, and Orc6 are tightly packed at the bottom lobe and as such their boundaries are speculative. (D) Docking with the highly curved archaeal Orc1/Cdc6 crystal structure (PDB id 2qby) as individual rigid body for the segmented Orc1-5 densities (). Archaeal Orc1/Cdc6 crystal structure in linear form (PDB id 1fnn) was docked as a rigid body into the segmented yeast Cdc6 density (). No Orc6 homolog crystal structure was found and its density was left undocked. The black double arrow in the front view indicates the distance between the putative Orc4 arginine finger and the Orc1 nucleotide-binding site. See also for how the rigid-body docked homolog crystal structure fits the segmented individual subunits Orc1, Orc4, Orc5, and Cdc6.

(A) Individual subunits are pulled apart to reveal their C-shaped structures of Orc1 through Orc5. Cdc6 density is extended. Assuming the yeast ORC subunits bind replication DNA in a similar manner, i.e. each subunit bind DNA with two claws, the origin DNA should follow a highly curved path lining the inner surface of the large crescent-shaped structure of ORC. The DNA path is illustrated by the dashed orange band. (B) An atomic model built with 72-bp dsDNA nested at the inner surface of the crescent-like ORC structure. Such DNA recognition model satisfies the general DNA interaction mode, and predicts a highly bent origin DNA onto which the MCM2-7 helicase will be loaded in the following steps. See also . See also for the architectural similarity between ScORC and DmORC.