3D reconstruction of the Mu A protein monomer. (A) Representative STEM dark-field single-particle images of the Mu A protein monomer. The top row shows the single-particle images cropped from the original STEM micrographs and low-pass filtered to enhance visibility in this photograph. The bottom row shows the corresponding images in the top row contrasted and segmented from the surrounding background signal. (B) 3D reconstruction of the Mu A monomer using 5408 molecular images, filtered to 16-Å resolution. The front view and rotations of 90° and 180° around the vertical axis are shown. The three protrusions in the head region of the structure are designated as P1, P2, and P3. (C) Front view in parallel stereo of the 3D reconstruction of the Mu A monomer.

3D reconstruction of the path of the DNA within the Mu Type 1 transpososome. (A) The transpososome is presented as a wire-mesh contour at the theoretical volume of the combined protein and DNA components. The DNA-phosphorus path (in gray) derived by electron spectroscopic imaging (ESI) is displayed in the complex at the same orientations as in Figure 3B and C, with numbers designating corresponding subunits. (B) Modeled DNA paths within the transpososome represented by a pair of curved rods with a diameter of 20 Å and a length of ∼170 Å. This DNA model was based on the phosphorus distribution from ESI and the modeling of the Mu A monomers into the transpososome structure (see text and Fig. 5). Each of the DNA rods contains the R1- and R2-binding sites and an extension of 5 bp at the R1 end representing the DNA between the R1 site and the Mu-terminal cleavage point (white portion ∼20 Å long at the bottom end of the ESI signal in the front and 90° views). The DNA portion at the top end in the same views is depicted in light gray in recognition of the weak ESI signal, potentially due to the flexible DNA end. The arrow shown in the front view indicates the position of the R1–R2 boundary.

Structural modeling of the Mu A protein monomer. (A) Biochemical domain structure of the 663-amino acid Mu A protein (see Chaconas and Harshey 2002 and Supplementary Legend for Fig. 6 for a detailed legend). Subdomain Iα, Iβ, Iγ, domain II, and domain III are depicted in purple, red, pink, blue, and green, respectively. Numbers above the boxes refer to amino acid positions at the beginning of each functional region. (B) Modeled DNA substrate (curved rod) bound by a Mu A monomer. The DNA rod contains the R1-binding site (in gray with a length of ∼80 Å) and the 5-bp DNA between the R1 site and the Mu-terminal cleavage point (in white with a length of ∼20 Å). The front view, top view, and 90° view in parallel stereo are shown. The DDE motif in the 90° view is represented by a yellow ball structure for D336, the only DDE residue at the surface of the 3D reconstruction as fitted with the aligned atomic structure of domain II (see text and C). The DNA in the R2-binding site follows a similar path on a corresponding Mu A monomer. (C) Docking the known atomic domain structures into the EM 3D reconstruction of Mu A. The color scheme for domains is the same as in A. The views at which the 3D EM structure (transparent yellow) is displayed are the same as in B. The N and C termini of the atomic ribbon structures are represented as black and green spheres, respectively. The extra mass in the fitted EM reconstruction is assigned to domain III (green ellipses). The DNA recognition helices of subdomain Iα, Iβ, and Iγ are depicted in brown. The residues of the DDE motif (D269, D336, and E392) are yellow ball-stick representations.

Mu transpososome assembly. (A) Schematic of the Mu strand-transfer reaction (see Chaconas and Harshey 2002 and Supplementary Legend for Fig. 1 for a detailed legend). (B) Type 1 complex formation under simplified reaction conditions with a pair of identical precleaved linear DNA substrates (50 bp) containing the Mu A protein R1- and R2-binding sites (Savilahti et al. 1995). In the presence of EDTA, Type 1 complex is formed and the reaction cannot proceed beyond this point. Black dots indicate the cleavage point at the Mu termini. This Type 1 transpososome was used for our EM studies. (C) Gel electrophoresis of the Type 1 transpososome used for our EM studies. Electrophoresis was performed on a 2% Metaphor agarose gel, which was subsequently stained with SYBR gold. Lane 1 contains the DNA substrate and lane 2 contains the substrate incubated with Mu A protein. The band above the tetrameric complex contains additional unstably associated Mu A molecules (Wu and Chaconas 1997). Tetrameric complexes were selected for structural analysis based on a mass calculation during image processing of the electron micrographs.

3D reconstruction of the Mu Type 1 transpososome. (A) Representative STEM dark-field single-particle images of the Mu Type 1 transpososome. The top row shows the single-particle images cropped from the original STEM micrographs and low-pass filtered. The bottom row shows the corresponding images contrasted and segmented from the surrounding background signal. (B) 3D reconstruction of the Mu Type 1 transpososome using 1912 molecular images, displayed at the theoretical molecular volume of the transpososome, corrected for DNA and protein densities. The front view and a 90° rotation around the vertical axis are shown. The orientation of the axis about which the structure displays twofold rotational symmetry (C₂) is shown in red. Each of the four subunits is designated by a number. In the front view, the flat (slightly concave) face of subunit 3 and the convex face of subunit 2 are labeled F and C, respectively. Images were filtered to 20 Å to facilitate interpretation of fitting of Mu A monomers (Fig. 2) and DNA-phosphorus in subsequent figures. (C) Parallel stereo top view of the 3D reconstruction of the Mu Type 1 transpososome at 90° rotation from the front view around the horizontal axis, at a slightly higher mass-density threshold (smaller volume) than in B, showing a big central cleft in the transpososome structure, which has the appearance of a tunnel in the front view of B.

Structural modeling of the Mu Type 1 transpososome. Four Mu A monomer 3D reconstructions filtered to 12.5-Å resolution (solid surface) were docked into the transpososome complex (wire-mesh contour at 20-Å resolution; see Supplemental Material, 3D reconstructions). The two blue monomers bind to one DNA molecule, and the two pink monomers bind to the other. The two monomers with darker color bind to the R2 sites, while the lighter colored ones bind to the R1 sites. The pair of DNA paths within the transpososome is shown in gray and white (see text). Subunit number designation and the symmetry axis orientations are as in Figure 3B and C.

Extended structural modeling of the Mu Type 1 transpososome. (A) The Mu Type 1 transpososome model with target and vector (donor) DNA. A 35-bp target DNA (orange double-stranded helix in standard B form) and the vector DNA beyond the Mu–host junction (in light gray with a length of ∼80 Å) have been added to the structure in Figure 5. The two catalytic DDE motifs are represented as two yellow ball structures. The R1–R2 boundary and point at which the L1–L2 loop would be extruded is indicated by the arrow on the left side of the front view. Note that in relation to this structure and those in B and C, the schematics in Figure 1A are upside down. (B) The 90° rotation of the model is displayed with the darker pink subunit blanked. (C) The 140° rotation of the front view in A around the vertical axis is displayed in parallel stereo. (D) Structural transitions of the Mu transpososome. This model simulates subunits 2 and 4 in the front view of A. The rotational movements of the subunits are indicated by the red arrows when the transpososome transits to the catalytically active configuration. The two catalytic DDE motifs are represented as two yellow balls and are brought into proximity of the scissile phosphates through the combination of the rotational displacement and a scissors-like movement.