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Figure 1 (A) Schematic diagram of the reaction mechanism of a 2-oxo acid dehydrogenase multienzyme complex. R is CH3 in the case of the PDH complex; ThDP, thiamin diphosphate; Lip, lipoic acid. (B) Schematic illustration of the three domains of the E2 chain that are connected by flexible linkers. (C) Atomic model for the B.stearothermophilus E2 catalytic domain monomer (Izard et al., 1999), and (D) schematic view of the trimer as proposed by Mattevi et al. (1992). Residues 184−200 form an extended, hook-like segment that was thought to stabilize the packing of the trimers. This interpretation is inconsistent with our findings for the size of the E1E2 complex (for details, see the text).
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 | Figure 2 (A) An image recorded from a frozen−hydrated specimen of the icosahedral E2 inner core consisting of 60 acetyltransferase domains. The scale bar represents 1000 Å. (B) Plot of the FSC (triangles) and the corresponding FSPR (circles) at different resolutions. The values in the plot reflect the resolution-dependence of the agreement between two halves of the set of 4458 molecular images used to construct the refined model. The resolution ( 14.5 Å) at which the FSC drops to 0.5 and the FSPR increases to 45° is conventionally taken to represent the resolution limit of the reconstruction. (C) Surface representation of the refined model for the E2 catalytic domain complex as viewed from the 3-fold axis. (D) Superposition of the model derived from our electron microscopic analysis with the atomic model obtained by Izard et al. (1999), based on X-ray crystallographic analyses from 3D crystals of the E2 catalytic domain complex. The docking was carried out manually in the crystallographic program O (Jones et al., 1991). The complex is 225 Å in diameter.
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Figure 3 (A) Electron micrograph recorded from a frozen−hydrated specimen of the E1E2 complex. The scale bar represents 2000 Å. (B) Gallery of molecular images of the E1E2 complex after filtering to suppress noise in the image, and inverting the density as an initial correction for the effects of the microscope CTF. Class averaged views (C) of the set of molecular images, two-dimensional projections (D) of the initial 3D model, and two- dimensional projections (E) of the refined 3D model, each shown in about the same orientation as the corresponding molecular images in (B). (F) A central slab of density from the refined 3D model of the E1E2 complex, which is 475 Å in diameter. The section shows that the structure of the E2 core is correctly recovered in the model of the E1E2 complex. (G) Plot of the FSC (triangles) and the corresponding FSPR (circles) at different resolutions.
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 | Figure 4 Stereo view of a surface-rendered representation of the refined 3D model of the E1E2 complex.
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Figure 5 (A and B) Views from the 3- and 5-fold axes of the manual fit of atomic coordinates of the backbone C atoms of a single E1 2 2 tetramer into the density of the 3D map of the E1E2 complex generated by electron microscopy. The E2 peripheral subunit-binding domains are shown in red. (C) Sectioned view of the density for the E1E2 complex with 60 manually-docked copies each of the atomic coordinates of the E1 22 tetramer, the E2 catalytic domain and the E2 peripheral subunit-binding domain. Sixty radial spokes have been included to illustrate the probable location of the linker domain connecting the peripheral subunit-binding and catalytic domains of the E2 chain.
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 | Figure 6 Contour plot of the core-weighted correlation function describing the locations of the two best fits of E1 to the outer density, based on the automated Monte Carlo search performed as described in the text. X' corresponds to the net spatial translation (x, y, z) from the best to the second-best fit, while  ' corresponds to the net 3D rotation (,  ,  ) angle from the best fit (global minimum) to the second-best fit.
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Figure 7 View of the docked E1 molecules (in yellow) and E2 peripheral subunit-binding domains (in red) from 3-fold (A and C) and 5-fold (B and D) vertices. The views in (A) and (B) are for the best fit obtained by automated docking, while the views in (C) and (D) are for the second-best fit.
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 | Figure 8 Model for active-site coupling in the E1E2 complex. Three E1 tetramers (in purple) are shown located above the corresponding trimer of E2 catalytic domains in the icosahedral core. Three full-length E2 molecules are shown, colored red, green and yellow, respectively. The lipoyl domain of each E2 molecule shuttles between the active sites of E1 and those of E2. The lipoyl domain of the red E2 is shown attached to an E1 active site. The yellow and green lipoyl domains of the other E2 molecules are shown in intermediate positions in the annular region between the core and the outer E1 layer. Selected E1 and E2 active sites are shown as white ovals, although the lipoyl domain can reach additional sites in the complex (for details, see text).
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Figure 9 View of the complex from one of the outer 3-fold vertices, showing multiple copies of the E1 22 tetramer. A lipoyl domain originating from one of the three E2 molecules at the inner 3-fold vertex can potentially reach the active sites of 9 E1 tetramers, which are located in the immediate vicinity (colored yellow), <120 Å (colored blue) or <140 Å (colored green) away from the pivot point of the swinging arm.
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 | Figure 10 Stereo view of an atomic representation of the complete E1E2 complex. The E2 catalytic and peripheral subunit-binding domains as well as the E1 22 tetramers are shown. The positions of the E1 22 tetramers and the peripheral subunit-binding domains are from the best fit identified by the automated procedure. For visual clarity, only the back half of the model is presented. The E2 catalytic domains, the peripheral subunit-binding domains and the E1 tetramers are colored green, red and purple, respectively.
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