The CMG helicase is composed of Cdc45, Mcm2–7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7–4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2–7, braced by Cdc45–GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2–7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.
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Cryo-EM data were collected on a Titan Krios I at the Howard Hughes Medical Institute, Janelia Farm. We also collected a cryo-EM data set on an FEI Polara with a K2 detector at the University of Texas Health Science Center. We thank the staff at these facilities for help with data collection. We also thank L. Pellegrini (University of Cambridge) for sharing the structure of human Cdc45 before publication. This work was funded by the US National Institutes of Health (GM111472 and OD12272 to H.L. and GM115809 to M.E.O'D.) and the Howard Hughes Medical Institute (M.E.O'D.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 3D classification procedure used to derive the 3.7-Å 3D density map of the Cdc45–GINS–Mcm2–7 NTD ‘platform’.
966,957 raw particles were selected from drift corrected electron micrographs. 2D classification and sorting rejected ~ 30% of the particles. 3D classification into six groups resulted in three similar 3D maps of the expected shape from our previous 15 Å CMG structure (Sun, J. et al. Nature structural & molecular biology. 12, 976-982, 2015), and the other three maps were either partial structures or distorted. Refinement using the ~470,000 particles of the three similar 3D maps led to a 3.8 Å 3D map. However, the density of the Mcm2-7 CTD motor region was noisy and indistinct. Excluding the CTD ring, the remaining region composed of Cdc45-GINS-Mcm2-7 NTD ring had an estimated resolution of 3.7 Å, based on the gold standard Fourier shell correlation curve.
Supplementary Figure 2 Euler-angle distribution of ~470,000 particles used in refinement of the 3.7-Å 3D map, and structure validation.
(a) The Thon rings in the power spectra of a typical drift-corrected electron micrograph reached to 3.0 Å. The lower left quadrant is a calculated version using the same CTF parameters as found in the experimental image. (b) The particle orientation covers all angular space. (c) Fourier shell correlations of the atomic model with the full 3D map (black), and the correlations of the 0.1-Å randomized and refined model against half map 1 (red), and with half map 2 (green), respectively. The 3.7 Å atomic model was randomly displaced by 0.1 Å. The noise-added model was refined by one round of coordinate and one round of b-factor refinement against half map 1. The refined coordinates were used to calculate FSC with half map 1, half map 2 and the full map respectively. The similarity between these curves indicates the atomic model is not over refined.
Supplementary Figure 3 Goodness of fit between the atomic model and the 3.7-Å density map of the Cdc45–GINS–Mcm2–7 NTD platform at selected subunit-interface regions.
(a) The 3D density map in blue mesh is superimposed with the atomic model in cartoon. Seven subunit interface regions were selected and magnified views are shown for the interfaces between: (I) Mcm4 and Mcm7, (II) Mcm3 and psf3, (III) Psf2 and Sld5, (IV) Mcm5 and Cdc45, (V) Cdc45 and Mcm2, (VI) Mcm2 and Mcm 6, and (VII) Mcm6 and Mcm4. (b) Five selected a-helices from Cdc45 and GINS showing the side chain densities of some of the relatively large residues.
Two rounds of focused 3D classification in the Mcm2-7 CTD motor ring region led to the identification of the 4.8-Å map with a tilted CTD-tier ring and the 4.7-Å 3D map with a untilted CTD-tier ring. The two red lines superimposed on the Mcm2-7 NTD-tier and CTD-tier densities highlight the distinct configuration of the two conformers.
(a) Surface rendered top, front side, and right side views of the 3D map of conformer I. (b) The gold standard Fourier shell correlation suggests a resolution of 4.8 Å at the 0.143 correlation point. (c) Color-coded resolution map of conformer I. (d) Surface rendered 3D map of conformer II. (e) Conformer II has an estimated resolution of 4.7 Å at the 0.143 correlation value. (f) Color-coded resolution map of conformer II. The early drop off of the FSC curves in (b) and (e) is reflected in the pinkish color of the resolution maps, particularly at the CTD motor region. This indicates that particles assigned to each conformer still contain a certain level of conformation heterogeneity.
Supplementary Figure 6 Superimposition of electron density and structural models for the CTD rings of the two CMG conformers.
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Supplementary Figure 7 Cross-links between Mcm5 WHD and the CTD-ring interior channel (top) and the surface charge of the CMG helicase (bottom).
(a) Back side view of CMG structure with the front Mcm2 and Mcm6 removed to better display the axial channel, as highlighted with two dashed black curves. (b) An enlarged view of the left boxed area in (a) showing the two crosslinks between K750 of the Mcm5 WHD and K477 in the Mcm3 AAA+ region and K534 in the Mcm5 AAA+ region, respectively. These crosslinks were observed in the complex of CMG helicase and the leading strand polymerase e. (c) The Mcm5 WHD structure is superimposed on the electron density in the region marked by the right box in (a). The residue that crosslinks with the CTD AAA+ ring interior channel is shown in red stick representation. (d) The CTD top view. The dashed turquoise circle marks the positively charged DNA entrance. (e) CMG side view. Two dashed turquoise lines mark a narrow positive band on the outside surface of Mcm3. (f-g) Cut-open back side and front side views of CMG surface charge. The dashed turquoise squares mark the positively charged interior channel surface of the OB fold region in the NTD ring. Dashed yellow lines mark the possible DNA path inside the channel.
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 2745 kb)
The video starts with an N-face view of conformer I, rotates it 90o to a back side view, then rotates it 90o to a front side view and morphs conformer I with conformer II. Then CMG conformer I is rotated 90o to a side view in which the GINSCdc45 project toward the viewer, and morphs it with conformer II. (MOV 24867 kb)
The Mcm5,2,6,4 subunits are colored yellow, blue, red, and green, respectively, and other subunits of CMG are removed for clarity. (MOV 22371 kb)
An animation illustrating the proposed DNA unwinding and translocation mechanism of the CMG helicase, showing the side view of a ratcheting CMG acting as a nodding pumpjack while traveling from left to right on a horizontal DNA. (MOV 16979 kb)
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Yuan, Z., Bai, L., Sun, J. et al. Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation. Nat Struct Mol Biol 23, 217–224 (2016). https://doi.org/10.1038/nsmb.3170
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