PMC

2D difference images from EM data show the position of the HsdR in the EcoR124I complex. (A) Difference imaging between image averages of large (left) and small (right) particles in the EcoR124I+DNA negative stain EM data set reveals a large “negative density” region (red contour at −2.5 σ), consistent with a missing HsdR in the small particles. (B) Difference imaging of HsdR in the open state of EcoR124I (without DNA). Although the relative flexibility of the open complex gives rise to a less well-defined difference map, a region of negative density consistent with a missing HsdR is visible nevertheless (red contour).

Gallery of type I RM structures and conformations determined by EM and single-particle analysis. (A) EcoR124I+DNA (closed state) negative stain EM. (B) EcoR124I without DNA (open state) negative stain EM. (C) EcoKI+DNA negative stain EM. For each 3 × 3 panel, the top rows are image averages, the middle rows are their corresponding reprojections, and the bottom rows are 3D surface views of the 3D reconstruction (bars, 200 Å); on the right is a larger 3D surface perspective view. Supporting EM data can be found in Supplemental Figure S2. Data on the assembly of the EcoR124I enzyme can be found in Supplemental Figure S3.

SANS and SAXS analyses. (A) SANS profiles of EcoR124I. The left panel shows the scattering data, and the right panel shows the pair distribution functions, p(r). (Gray) Protonated EcoR124I in 0% D₂O; (blue) MTase core in situ within the RM enzyme (deuterated HsdR and protonated MTase measured in 100% D₂O); (red) the two HsdR in situ in the RM enzyme (deuterated HsdR and protonated MTase measured in 40% D₂O). (B) SAXS profiles of EcoR124I (black) and EcoKI (green). The panel on the left shows the scattering data, and the right panel shows the pair distribution functions, p(r). In both A and B, the solid lines in the scattering data represent the fits from the corresponding back-transformed distance distribution functions, p(r), in the panel on the right. (C) Multiphase ab initio modeling showing the location of the MTase core (blue) and the HsdR (red), superimposed on the EM map of EcoR124I from (gray). The panel on the right shows a 90° rotation about the long axis in the left panel. Data on the assembly of the EcoR124I enzyme can be found in Supplemental Figure S3.

2D and 3D difference mapping from EM data shows the route of DNA/Ocr through the EcoR124I complex. (A) Negative stain EM difference image averages of EcoR124I+DNA and EcoR124I+Ocr from two orientations show a smaller central area of positive difference (green contours at +4.5 σ), indicating the position of the DNA mimic Ocr, which excludes stain more effectively than DNA. (B) Surface view of the 3D reconstruction of EcoR124I+DNA. (C) Surface view of the 3D reconstruction of EcoR124I+Ocr shows the central hole is mostly occluded in the Ocr-containing complex when compared with the EcoR124I+DNA surface shown in B. (D) Two views of the DNA/Ocr 3D difference map (green surface, contoured at +4.5 σ) overlaid onto the EcoR124I+DNA map (gray mesh) showing the main positive difference densities. (E) Two views of the EcoR124I+DNA 3D map (gray mesh) with the EcoKI MTase core+Ocr atomic model (PDB code: 2y7C) docked in as a single rigid body. (Magenta spacefill) Ocr; (yellow ribbon) HsdS; (blue ribbon) 2× HsdM. The path of Ocr, as predicted from the MTase structure, matches well to the difference density.

Schematic of large-scale conformational change and initiation of DNA looping and translocation. (A) Type I RM enzymes exist in a dynamic equilibrium between open and closed states (movement is shown by orange arrows, and pivot points in C-terminal regions of HsdM are indicated by pink dots). DNA (green) binding to form encounter complexes can occur nonspecifically to the HsdR (red) or via the target sequence to the MTase core (HsdM is in light and dark blue, and HsdS is in yellow). Complete closure of the enzyme and bending of the DNA around the HsdR produces the initiation complex for DNA translocation. (B) The predicted complete path of the DNA (green dots) through the atomic model of EcoR124I with segments of bound DNA. This is the proposed initiation complex (from ). During active translocation, the DNA would then form expanding loops from each side (light-green dots for DNA, and the direction of translocation is shown by black arrows). The inset shows the initiation complex turned 90° to the main panel.

Structural evolution of type IIG RM enzymes from a type I RM enzyme undergoing fusion of the C terminus of an endonuclease domain from HsdR, via deletion of the motor domains, to the N terminus of HsdM. The structure on the left shows part of EcoR124I, with one endonuclease domain from HsdR (in red), one HsdM (N-terminal domain is in green, and the MTase catalytic domain is in blue), and the HsdS (in yellow) (two TRDs). DNA bound to the MTase core is shown, but DNA bound to HsdR is omitted for clarity. The dashed line shows how the end of the endonuclease domain could join with the N terminus of HsdM to form a structure similar to the type IIG structures shown on the right. The catalytic motifs in the endonuclease domain and HsdM are shown in spacefill. The middle structure shows the structural model of MmeI with bound DNA with the same coloring used for equivalent domains (endonuclease domain, N-terminal domain, MTase catalytic domain, and TRD) (; coordinates from ftp://genesilico.pl/iamb/models/RM.MmeI). The structure on the right shows the crystallographic structure of BpuSI (PDB code: 3s1s) with the same coloring of domains as in the other structures and with an inserted extra domain shown in gray (). DNA is absent in this structure, and one can see that the endonuclease domain would be blocking the DNA-binding site on the TRD. proposed that the endonuclease domain would twist away to allow DNA sequence recognition.

Atomic models of EcoR124I+DNA, EcoR124I, and EcoKI+DNA docked into the EM map densities. (A) Two views of the EcoR124I+DNA model showing the MTase core closed around DNA (green; DNA bound to each HsdR is not shown for clarity). Adenine bases are flipped out into the active sites of each of the two HsdM (light and dark blue), induced by an ∼45° bend in the DNA. The HsdS is in yellow, and the two HsdR are shown in red, with the β sheets of the recA-like motor domains colored orange. Residues missing from the crystal structures (the 44 and 152 C-terminal residues of HsdM and HsdR, respectively) were modeled de novo and are shown in gray. The C-terminal regions of HsdM extend down to bind at the coiled coil of HsdS, and the HsdR C-terminal domains fill some empty density next to the N terminus of HsdM. (B) A model for the second type I RM enzyme, EcoKI bound to DNA. Colors are as in A, with residues modeled de novo shown in gray. The HsdS and HsdM from the MTase structure (PDB code: 2y2C) were docked in as a single rigid body. The HsdR modeled on those from EcoR124I (PDB code: 2w00), as described in the Supplemental Material, were placed in a position analogous to the EcoR124I model. (C) The model of EcoR124I in the open conformation (i.e., without DNA). Colors are as in A, with residues modeled de novo shown in gray. Although the EM map is at a lower resolution, a full atomic model can be built, aided by the EcoR124I+DNA model, SANS data, and 2D difference imaging. The HsdM and HsdR swing out as a unit away from HsdS. The predicted hinge regions in the C termini of the HsdM (modeled in gray) and their connections to HsdS are not well resolved.