Fig. 1: In vitro assembly and cryo-EM analysis of the T4 primosome.

a Domain architecture of the gp41 helicase, the gp61 primase, and the ssDNA/RNA primer construct used in the in vitro assembling of the primosome. b Step-by-step process used to assemble the T4 primosome. c 3D EM maps of the step 1 complex—(I) the hexameric gp41 helicase in an open spiral configuration; two step 2 complexes—the ssDNA-bound gp41 helicase in the (I) open spiral and (II) planar closed-ring configurations; and two step 3 complexes—(I) the ssDNA-bound gp41 helicase in an open spiral form and (II) the primosome. The average resolution of each map is labeled below the respective map. The primosome map (3-II) is a composite map of the separately refined maps of the ssDNA-bound gp41 helicase (2.7 Å) and the gp61 primase region of ssDNA-bound gp41 helicase-gp61 primase complex. The overall resolution of the composite primosome map reaches 3.2 Å, but the resolution of the primase region is lower; see the detailed local resolution estimation in Supplementary Fig. 5. The EM maps are postprocessed by DeepEMhancer (v0.14, using the “tightTarget” model) and aligned in such way that the gap in open spirals or the seam in closed rings face the readers. The two open-spiral, ssDNA-bound gp41 helicase structures (2-I and 3-I) are the same; and the closed-ring gp41 helicase structure (2-II) is essentially the same as the gp41 helicase region in the primosome structure (3-II). Created with BioRender.com.