Fig. 3: Use of maps where parts of the relaxosome have been deleted to generate the complete structure of the relaxosome.
a Superposition of the unsharpened global maps of ss-27_+8ds+9_+143-R (in light blue mesh) and ss-27_+8ds+9_+143-RΔAH+CTD (in light red surface). Maps details are reported in Supplementary Fig. 4h and i, respectively. A representative 2D class is shown on the left. The structure of ss-27_+8ds+9_+143-R is in dark grey ribbon. A model of TraI AH and CTD (in violet ribbon) is shown fitted in the additional density observed in ss+8_-27ds+9_+143-R, but not in ss+8_-27ds+9_+143-RΔAH+CTD. Two orientations, 90° apart, are shown at left (same orientation in inset) and right. The lack of resolution for TraI AH and CTD is not surprising. Firstly, TraI is a remarkably flexible protein. It unfolds readily to pass through the pilus during conjugative transfer and it is known to be unusually susceptible to mild-proteolytic cleavage either in its unbound form or bound to its TE ssDNA-binding site (traITE in Fig. 1b)14,77. It is known to be ordered only in its helicase-binding mode14,78. Moreover, both TraI AH and CTD together with TraM are located in a region of the DNA that is itself ill-defined, indicating flexibility of the DNA in this region. b Superposition of the unsharpened global maps of ss-27_+8ds+9_+143-R (in light blue mesh) and ss-27_+8ds+9_+143-RΔTraM (in light green surface). Map details are reported in Supplementary Fig. 4h,j, respectively. A representative 2D class is shown on the left. The structure of ss-27_+8ds+9_+143-R is in dark grey ribbon. A model of TraM (in magenta ribbon) is shown docked in the additional density observed in ss+8_-27ds+9_+143-R but not in ss+8_-27ds+9_+143-RΔTraM. Two orientations, 90° apart, are shown at left (same orientation in inset) and right. To validate the TraM location, we also generated an extended model of the DNA that includes sbmC and observed that sbmC locates in the density region attributed to TraM. Moreover, the molecular interactions of TraM within this region of DNA are very similar to those identified previously by hydroxyl radical foot printing thereby providing another layer of confidence that the density for TraM has been properly ascribed (Fig. 5d and Supplementary Fig. 9c)20.
