Volume 16, Issue 9, Pages (September 2008)

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Volume 16, Issue 9, Pages 1428-1436 (September 2008) Quaternary Structure of Flavorubredoxin as Revealed by Synchrotron Radiation Small- Angle X-Ray Scattering  Maxim V. Petoukhov, João B. Vicente, Peter B. Crowley, Maria Arménia Carrondo, Miguel Teixeira, Dmitri I. Svergun  Structure  Volume 16, Issue 9, Pages 1428-1436 (September 2008) DOI: 10.1016/j.str.2008.06.009 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 FDP Organization and Electron Transfer (A) Scheme depicting the electron transfer chains in E. coli and D. gigas involving FDPs as nitric oxide and/or oxygen reductases. Fe-Fe box, metallo-β-lactamase domain of FDP; FMN box, flavodoxin domain of FDP; Rd box, rubredoxin, an additional module of E. coli FDP (flavorubredoxin, FlRd), or a redox partner of D. gigas FDP; FAD box, NADH:(flavo)rubredoxin oxidoreductase. (B) The tetrameric assembly of FDPs as revealed by X-ray crystallography. Note the dimer of dimers arrangement. The chains from one of the dimers are highlighted in dark and light blue with the diiron and FMN groups shown as spheres. The other dimer is colored orange and yellow. The coordinates of M. thermoacetica FDP (PDB ID code 1YCH; Silaghi-Dumitrescu et al., 2003) were used to prepare the figure in PyMOL (DeLano, 2002). Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Scattering Patterns and Distribution Functions (A) SAXS data of FlRd constructs. (1) Rd module, (2) FDP core, and (3) full-length FlRd. Experimental data are denoted by dots, and fits from ab initio and rigid-body models (homology model for Rd domain) are shown as blue solid and red dashed lines, respectively. The fit from the crystallographic tetramer (Figure 1B) of M. thermoacetica FDP is given as a green dashed line. (B) Distance distribution functions of the FDP core (red) and full-length FlRd (blue). Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Ab Initio Modeling of FlRd Constructs (A) Ab initio models of FlRd (gray beads) and the FDP core (blue beads) reconstructed in P222. (B) Comparison of the ab initio shape of FDP with the crystallographic tetramer of of M. thermoacetica FDP (shown as a red backbone trace). Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Rigid-Body Models of FlRd (A) Reconstruction in P222. (B) No symmetry restraints for the Rd domain and linkers. (C) P222 modeling with the requirement of contacts between Rd domains. These are displayed in space-filled mode with the same color scheme for the FDP tetramer as in Figure 1B. The linkers and Rd domain are colored green and magenta, respectively. The middle and bottom views are rotated by 90° about the horizontal and vertical axes, respectively. Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 The Distributions of the Radii of Gyration in the Ensembles of FlRd Pools containing the FDP core with four Rd domains attached by random linkers and consisting of 10,000 asymmetric structures (1), 1,000 symmetric structures (2), and 1,000 asymmetric models with extended linkers (3). Distribution (4) corresponds to the average over 150 ensembles selected by EOM. Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 6 EOM Representative Models A typical ensemble selected by EOM from a pool of randomized structures. The bottom view is rotated by 90° about horizontal axis. Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 7 Comparison of the Experimental Scattering Profiles of Dg_ROO and FlRd The scattering curve computed from the crystallographic Dg_ROO tetramer (PDB ID code 1YCH) is presented as a green solid line. Dg_ROO, red circles; FlRd, blue triangles. Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 8 Electrostatic Potentials of FDP and Rd Structural models of (A) the FDP core and (B) the Rd module of FlRd, built in SWISS-MODEL (Arnold et al., 2006) using the structures of M. thermoacetica FDP (PDB ID code 1YCH; Silaghi-Dumitrescu et al., 2003) and C. pasteurianum rubredoxin (PDB ID code 4RXN; (Watenpaugh et al., 1980). The dashed circles indicate the approximate location of the FMN and diiron cofactors in FDP. The surface maps were generated using PyMOL (DeLano, 2002). Structure 2008 16, 1428-1436DOI: (10.1016/j.str.2008.06.009) Copyright © 2008 Elsevier Ltd Terms and Conditions