(a) Ribbon diagram of the motor module resembling a cashew nut, with ORC1 in green, ORC4 in cyan and ORC5 in purple. The three ATP molecules nestled between the domains are shown in stick …
Bar diagram illustrating the constructs and domain structure of HsORC subunits. The colored regions were used in this study. For ORC5, the construct used for the motor module extends till residue …
SigmaA-weighted omit maps of three of the six ATP nucleotides at the three different interfaces from left to right: at the ORC1-4 interface, at the ORC4-5 interface, at ORC5 ATP-binding site.
Top row: HsORC1/4/5 (this work); yeast RFC-B, -C, -D, -E subunits with nucleotide from the RFC clamp loader bound to the PCNA clamp (Bowman et al., 2004) (1SXJ); gp44 subunits with nucleotide from …
(a) ATP-binding site at the ORC1/4 interface. (b) ATP-binding site at the ORC4/5 interface. (c) ATP-binding site of ORC5. ORC1 is shown in green, ORC4 in cyan and ORC5 in purple. ATP and key …
(a) ATP-binding site at the ORC1/4 interface. (b) ATP-binding site at the ORC4/5 interface. (c) ATP-binding site of ORC5. ORC1 is shown in green, ORC4 in cyan and ORC5 in purple. ATP and key …
Representative samples from ATPase assays. (a) Thin layer chromatography (TLC) of ATPase activity assays of the ORC motor module and mutants as indicated. (b) Coomassie-stained SDS-PAGE gel showing …
(a) Cryo-EM density of HsORC1-5, back view. Note that the top and bottom are flipped compared with the maps shown in the supplementary Figures. (b) Ribbon diagram of HsORC modeled into cryo-EM …
Electron-density map directly following molecular replacement using DmORC2/3 as a search model contoured at 1σ. This illustrates that even at this modest resolution the crystallographic data has …
(a) A typical raw micrograph of the HsORC particles stained in uranyl acetate. (b) 2D class averages. Numbers at the lower left corner in each panel refers to the number of particles used to compute …
(a) The 3D map in six different views. Note that in these views, the top and bottom are flipped (upside down) compared with Figure 4. (b) Gold standard Fourier Shell correlation of the reconstructed …
(a) Left panel, a typical raw image for initial manual particle picking. Right panel, around 100 particles marked by green circles were picked manually. (b) Left panel, a second raw image used for …
532,782 raw particles were selected from drift corrected electron micrographs. 2D classification and sorting rejected ~80% of particles that did not yield good averages or appeared to be broken …
Euler angle distribution of 10,357 particles used in refinement of the 20 Å 3D map. The particle orientation covers nearly all angular space.
(A) A typical motion-corrected raw image of HsORC particles frozen in vitreous ice recorded on a K2 direct detector. (B) Selected 20 2D class averages. (C) 3D cryo-EM map of HsORC, front, back, side …
Ribbon diagram of HsORC modeled into cryo-EM density in an orthogonal view to that shown in Figure 3b. The ORC motor module is colored as in Figure 1. ORC2 is shown in wheat and ORC3 is shown in …
(a) Different positions of HsORC1 and DmORC1 in the context of the bigger particle. HsORC1 is shown in green, DmORC1 is shown in red and the rest of the HsORC complex is shown in grey. (b) The …
(a) Model of HsORC-CDC6 with CDC6 in pink. HsORC1-5 subunits are shown in a transparent rendering. (b) Cartoon depiction of HsORC-CDC6 showing the organization of the particle with the smaller WHDs …
Side view of the model of HsORC-CDC6 with CDC6 in pink. CDC6 completes the ring without any additional movements.
Top view of the model of HsORC-CDC6 with DNA.
ORC1 is shown in green, ORC4 in cyan, ORC5 in purple, ORC2 in wheat and ORC3 in salmon. ATP is shown in stick.
HsORC1 moves to form an active ATP interface with HsORC4. HsORC2 moves out and up from the more ‘collapsed’ placement in DmORC.
Data collection and refinement statistics for HsORC motor module*.
Data collection | |
---|---|
Wavelength (Å) | 0.9793 |
Resolution range (Å) | 19.88–3.39 (3.52–3.39) |
Space group | P21 |
Unit cell (Å,°) | 120.89 81.14 151.95 90 97.25 90 |
Total reflections | 148448 (14543) |
Unique reflections | 39700 (3849 ) |
Multiplicity | 3.7 (3.8) |
Completeness (%) | 0.97 (0.96) |
Mean I/s(I) | 8.75 (1.19) |
Wilson B-factor (Å2) | 104.85 |
R-merge | 0.1385 (1.406) |
R-measure | 0.1619 (1.641) |
CC1/2 | 0.997 (0.524) |
CC* | 0.999 (0.829) |
Refinement | |
---|---|
Resolution range (Å) | 19.88–3.39 (3.52–3.39) |
Reflections used in refinement | 39700 (3849) |
Reflections used for R-free | 1963 (198) |
R-work | 0.2421 (0.3745) |
R-free | 0.2811 (0.4015) |
CC(work) | 0.959 (0.662) |
CC(free) | 0.923 (0.591) |
Number of non-hydrogen atoms | 15870 |
Macromolecules | 15678 |
Ligands | 192 |
Protein residues | 1947 |
RMSD bonds (Å) | 0.004 |
RMSD angles (°) | 0.72 |
Ramachandran favored (%) | 95 |
Ramachandran allowed (%) | 4.6 |
Ramachandran outliers (%) | 0 |
Rotamer outliers (%) | 0.57 |
Clashscore | 3.59 |
Average B-factor (Å2) | 127.05 |
Macromolecules | 127.23 |
Ligands | 112.92 |
Number of TLS groups | 31 |
*Values in parentheses are for the highest resolution shell.
Data collection and refinement statistics for HsORC2/3*.
Data collection | |
---|---|
Wavelength (Å) | 0.9793 |
Resolution range (Å) | 20.07–6.00 (6.21–6.00) |
Space group | P21 |
Unit cell (Å,°) | 87.26 114.96 316.46 90 90.72 90 |
Total reflections | 52200 (8302) |
Unique reflections | 15430 (2286) |
Multiplicity | 3.4 (3.6) |
Completeness (%) | 95.0 (99.1) |
Mean I/s(I) | 5.7 (1.0) |
Wilson B-factor (Å2) | 316 |
R-merge | 0.183 (>1) |
R-measure | 0.24 (>1) |
CC1/2 | 0.991 (0.422) |
Refinement | |
---|---|
Resolution range (Å) | 20.07–6.00 (6.45–6.00) |
Reflections used in refinement | 15179 (1498) |
Reflections used for R-free | 753 (1498) |
R-work | 0.3180 (0.3804) |
R-free | 0.3685 (0.4019) |
Number of non-hydrogen atoms | 24148 |
Protein residues | 2944 |
RMSD bonds (Å) | 0.013 |
RMSD angles (°) | 1.30 |
Ramachandran favored (%) | 87 |
Ramachandran allowed (%) | 12 |
Ramachandran outliers (%) | 0.8 |
*Values in parentheses are for the highest resolution shell.
Cryo-EM data collection and refinement statistics of HsORC1-5.
Data Collection | |
---|---|
EM equipment | FEI Titan Krios |
Voltage (kV) | 300 |
Detector | Gatan K2 |
Pixel size (Å) | 1.01 |
Electron dose (e-/Å2) | 50 |
Defocus range (µm) | −1.5 ~ −3.5 |
Reconstruction | |
---|---|
Software | RELION 1.4 |
Number of final particles Resolution (Å) | 10,357 20 |
Map sharpening B-factor (Å2) | −479 |