Cryo-EM reconstruction of the DNA-bound PolD binary complex.

(A) Backbone trace of the cryo-EM model of PolD. The blue region was built by fitting the DP1 H451A (PDB ID: 6HMF, this study) and DP2 (PDB ID: 5IJL [15]) crystal structures in the cryo-EM map. The C-terminal region of DP2 (1090–1195) (green) was built guided by the homology with the CTD of the catalytic subunit of human Polε (PDB ID: 5VBN [24]) and adjusted manually in the cryo-EM density. Part of the DPBB-1 domain and the DPBB-connecting loop (green) were built by homology modeling guided against the structure of yeast RNAP-II (PDB ID: 2E2I [25]). A 15-/16-mer B-form DNA was generated with Coot [26] (yellow) and rigid-body fitted into the cryo-EM density. Finally, two α helices (red) were built de novo in the cryo-EM density: α14 (324–338) and α43 (1074–1089)). (B) Enlarged view showing the α14 and α43 α helices that were built de novo. The cryo-EM map surrounding these helices is shown as a gray mesh contoured at 6 σ. (C) Secondary structure predictions of Jpred [27] for the de novo–built α14 and α43 α helices. The confidence factor is indicated: 0, not confident, and 9, very confident. (D) Detailed views of the DNA-bound PolD cryo-EM experimental map. Density surrounding PolD and DNA is shown in gray mesh contoured at 6 σ. Residual peaks of density are shown in red mesh contoured at 12 σ. cryo-EM, cryo–electron microscopy; CTD, C-terminal domain; DPBB, double-psi β-barrel; OB, oligonucleotide binding; PDB, Protein Data Bank; PDE, phosphodiesterase domain; RNAP, RNA polymerase.