Figure 5. Cryo-EM structure of Sen1^N-PP-C.

(A) Local resolution of Sen1^N-PP-C, calculated in using the Local Resolution Estimation in CryoSparc, and displayed using a blue (high-resolution, 3.17 Å) to red (low-resolution, 4.0 Å) color-coded surface.

(B) Three interaction interfaces (Int1-Int3) mediate Sen1^Sen1N interaction with Sen1^Hel. The LZZ from Sen1^Hel binds both Int1 and Int3 on Sen1^Sen1N. A ΔLZZ internal deletion construct is marked.

(C) Consurf analysis of the Sen1^Sen1N surface reveals conserved surface interaction interfaces (Int1-Int3, dotted lines) are involved in interactions with Sen1^Hel. Top: Sen1N domain interaction interfaces that bind the helicase domain. Bottom: Helicase domain interaction surfaces that bind the Sen1N domain.

(D) Molecular details of the Int1 interdomain Sen1^Sen1N - Sen1^Hel interaction interface.

(E) Cryo-EM density for Sen1^Sen1N Int2 region displayed contoured at 11.0 σ overlaid upon a molecular surface diagram of the β-barrel domain (orange).

(F) Molecular details of the Int3 interdomain Sen1^Sen1N - Sen1^Hel interaction interface.

(G) MBP-pulldowns show the LZZ is critical for the Sen1^Sen1N–Sen1^Hel interaction.

(H) Trans inhibition of Sen1^Hel by Sen1^Sen1N. Sen1^Hel (1 nM) was pre-incubated with Sen1^Sen1N at the indicated concentrations for 5 min on ice. Reactions were initiated by addition of 50 nM Sub7 (Supplementary Table 3) and a mixture containing ATP (1 mM) and MgCl₂ (2 mM). The percentage of unwound ssDNA was calculated from the FAM signal at 30 min. Error bars are SD from 3 replicates, **p<0.01, *p<0.05.