1. Volume 28, Issue 3, Pages 434-445 (November 2007)
Structural Aspects of RbfA Action during Small Ribosomal Subunit Assembly 
Partha P. Datta, Daniel N. Wilson, Masahito Kawazoe, Neil K. Swami, Tatsuya Kaminishi, Manjuli R. Sharma, Timothy M. Booth, Chie Takemoto, Paola Fucini, Shigeyuki Yokoyama, Rajendra K. Agrawal 
Molecular Cell 
Volume 28, Issue 3, Pages (November 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Crystal Structure of Tth RbfA and Its Comparison with Known Atomic Structures of RbfA from Other Species
(A) Stereo representation of the Tth RbfA is shown in cartoon (PDB ID, 2DYJ).
(B) An enlarged view of the helix-kink-helix motif (cyan) with all residues in stick. Residues involved in notable interactions, Asp25 and Arg27 on the 310 helix (magenta) and the conserved Phe87, are also shown in stick.
(C) Stereo representation of Tth RbfA in the surface potential prepared by using APBS tools built in PyMOL.
(D) Superposition of structures of RbfA from T. Thermophilus (molecules A and B in the asymmetric unit are shown in dark and light blue colors, respectively), H. influenzae (green, 1JOS), and M. pneumoniae (orange, 1PA4). In all panels, N, C, and hkh mean N terminus, C terminus, and helix-kink-helix motif, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Binding of RbfA to the 30S Ribosomal Subunit
(A) SDS-PAGE results showing the binding of RbfA to the 30S subunit. Tth 30S subunits were incubated alone (lanes 2–4), or with increasing concentrations of RbfA (10-fold [lanes 5–7], 20-fold [lanes 8–10], and 40-fold [lanes 11–13] molar excess over 30S subunit), before being centrifuged through a 10% sucrose cushion (see Experimental Procedures). As a control, reactions were also performed with RbfA in the absence of 30S subunits (lanes 15–20). For each condition, aliquots of the initial (pre) reaction, supernatant (S), and pellet (P) were subjected to 20% SDS-PAGE and stained with Coomassie blue. RbfA pellets only in the presence of the 30S subunit, and the stoichiometry of binding increases with increasing initial excess of RbfA over 30S subunit. Lanes 1 and 14 are marker lanes, with 14, 20, and 33 kDa bands indicated.
(B) Cryo-EM map showing extra mass of density that encompasses the binding position of RbfA (red) on the Tth 30S subunit (yellow).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Conformational Changes in the 30S Subunit upon RbfA Binding
A portion of the difference map (red) shown in Figure 2B corresponds to a large positional shift of the 3′ minor domain of the 16S rRNA involving the decoding site helix 44. Both positions of helix 44 (h44), as well as those of helix 45 (h45), are shown. Purple ribbons, original positions; red/orange ribbons, shifted positions. B2a and B3 indicate the positions of the intersubunit bridges. Arrow indicates direction of the shift. The landmarks of the 30S subunit are: h, head; p, platform; and sp, spur.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Localization of RbfA on the 30S Subunit, and Comparison of the Atomic Structure of RbfA with the Cryo-EM Density Map
(A) Interpretation of the extra mass in terms of 30S subunit conformational change, involving 16S rRNA helices 44 and 45 (orange), and RbfA mass (red). The mass attributable to RbfA was derived after subtraction of the density corresponding to shifted positions of helices 44 (h44′) and 45 (h45′) from the total extra mass shown in Figure 2B.
(B and C) Stereo representations of the fittings of (B) the X-ray crystallographic structure (CCF, 0.78) and (C) the homology model (CCF, 0.79) of the Tth RbfA into the corresponding cryo-EM density. The asterisk (∗) in (B) points to an unoccupied region of the cryo-EM density, due to absence of three amino acid residues and a different orientation of the tail in the X-ray structure. However, the same density region is nicely accounted for by the homology model (C). Thumbnails to the left of the panels depict the orientations of the 30S subunit, with body (b), head (h), and platform (p) identified.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Interactions of RbfA with 16S rRNA Helices 44 and 45
(A) Stereo-view presentation of the interaction between the hkh motif of RbfA (red) and the linker region between the shifted positions of helices 44 (h44′) and 45 (h45′). The hkh motif is shown in cyan.
(B) Correlation between the electrostatic distribution of RbfA (shown with regions of positive and negative potentials, in blue and red, respectively) and interaction of RbfA with h44 and h45. Both positions of h44 and h45 are shown: light purple ribbons, original positions; brown ribbons, shifted positions (marked as h44′ and h45′). (B) was made with PyMOL ( In both panels, the RbfA-30S complex is viewed from the platform side, as depicted in the thumbnail to the lower right.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Interactions of RbfA with Other Components of the 30S Subunit
(A) Proximity of the C terminus of RbfA (red) to helix 1, central pseudoknot helix 27, and helix 28 of the 16S rRNA.
(B) Other neighbors of RbfA within the subunit. Positions of 16S rRNA segments and r-proteins were defined by docking of the crystallographic structure (Wimberly et al., 2000) into the cryo-EM map. Numbers prefixed by h and S identify 16S rRNA helices and 30S small subunit proteins, respectively. The C terminus was positioned as in the Tth RbfA homology model because in the Tth RbfA crystallographic structure the C-terminal end would clash with the 16S rRNA. Thumbnails to the left depict the orientations of the 30S subunit.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Comparison of the Binding Positions of RbfA and Era on the 30S Subunit
(A) Binding position of RbfA (red) and Era (magenta; Sharma et al., 2005) on the 30S subunit.
(B) RbfA (red) and Era (magenta) interact with a common structural element, h28, of the 16S rRNA (cyan). The thumbnail to the left depicts the orientation of the 30S subunit.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions