1. Volume 15, Issue 12, Pages 1663-1673 (December 2007)
Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another Twist 
Susanna Törnroth-Horsefield, Pontus Gourdon, Rob Horsefield, Lars Brive, Natsuko Yamamoto, Hirotada Mori, Arjan Snijder, Richard Neutze 
Structure 
Volume 15, Issue 12, Pages (December 2007)
DOI: /j.str
Copyright © 2007 Elsevier Ltd Terms and Conditions

2. Figure 1 Structure of the AcrB:YajC Complex
(A and B) Overview of AcrB (orange) with an elongated, highly tilted single TM helix (green, assigned as YajC) in close association with the TM domain of AcrB. Ampicillin molecules are shown in blue space fill. Only the two associated with the front protomer are shown for clarity; however, there are six in total for the trimer, as shown in (B). See Figure S1A for the initial 2Fobs-Fcalc composite omit map for the novel TM helix following molecular replacement and simulating annealing prior to adding model. See Figure S1B for a 2Fobs-Fcalc map of a selected region of the TM domain of AcrB. (B) Top view of the TM region of AcrB (orange) with YajC (green) illustrating all six ampicillin molecules in the central cavity of the AcrB trimer.
(C) Excess 2Fobs-Fcalc electron density (blue mesh, contoured at 1.0 σ) assigned as ampicillin molecules. See Figure S7 for Fobs-Fcalc and 2Fobs-Fcalc composite omit maps for both ampicillins in stereo.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions

3. Figure 2 Structure of YajC and Its Region of Interaction with AcrB
(A) Stereo view of the 2Fobs-Fcalc electron density map (blue mesh, contoured at 1.0 σ) for the elongated, highly tilted single TM helix of YajC.
(B) Alternate stereo view of YajC (light green) showing five bulky side chains (dark green) assigned from the electron density maps; 2Fobs-Fcalc composite omit map (pink mesh, contoured at 1.0 σ) and Fobs-Fcalc map with positive peaks (black mesh, contoured at 2.8σ) and negative peaks (red mesh, contoured at −3σ) are shown. Both maps were calculated with a polyalanine model of YajC to identify the positions of the bulky side chains.
(C) YajC (green ribbon) binds to a region of AcrB (molecular surface, one monomer shown) composed of highly conserved residues. The surface of AcrB is colored according to sequence conservation (averaged over 5 Å in space for clarity), ranging from blue (low conservation) through white to red (high conservation). The view is similar to the one of Figure 1A.
(D) Close up of the AcrB:YajC interface. Conserved residues of YajC related members match the conserved surface of AcrB as shown in (C). YajC (ribbon and sticks) is colored from blue (low conservation) through white to red (high conservation), and the surface of AcrB is colored orange.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions

4. Figure 3 Comparison between YajC and the γ Subunit of SecY
(A) Overview of the SecY complex (pink, left) and TM domain of AcrB (orange, right) with YajC (green) highlighting the highly tilted nature of both the γ subunit of SecY (purple) and YajC in the membrane.
(B) Structural overlay of YajC and γ subunit of SecY showing a similar curvature/kink. Root mean square deviation for 30 Cα atoms is 0.96 Å.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions

5. Figure 4 Conformational Changes Occurring within the AcrB
(A) Structural perturbations in the AcrB:YajC complex relative to an isomorphous structure (PDB code: 1IWG) lacking the novel TM subunit. Structures were aligned upon the TM domains and are viewed perpendicular to the membrane from the cell exterior. Arrowheads represent the direction and relative magnitude of the observed movements where, for clarity, arrows are five times larger than the actual protein movement. An example arrowhead (top right) illustrates the scale of these movements. A rotation of the porter domain around the central symmetry axis is apparent. Figure S5 provides an identical comparison with other isomorphous structures of AcrB versus 1IWG.
(B) An identical representation to (A) but with both structures aligned upon the DN and DC domains of the TolC binding domain of AcrB.
(C) An identical representation as in (B) but comparing the structure of AcrB determined from an asymmetric space group (Murakami et al., 2006) (PDB code: 2DRD) against 1IWG. Figure S6 provides an identical comparison with other asymmetric structures of AcrB versus 1IWG.
(D) An identical representation of the putative conformational changes required to open the periplasmic entrance of TolC (gray). Arrows generated by comparing a model for the open conformation of TolC (Fernandez-Recio et al., 2004) against the X-ray structure of the closed conformation (Koronakis et al., 2000). All panels are drawn to the same scale.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions

6. Figure 5
Summary of the Growth of E. coli Deletion Strains in the Presence of Ampicillin and Nafcillin
(A) Relative optical densities 6 hr after inoculation for wild-type (WT, black), yajc (blue), and acrb (green) E. coli deletion strains in the presence of ampicillin. Ampicillin concentrations (0 mg/l, 5 mg/l, 10 mg/l, 15 mg/l, 20 mg/l, 30 mg/l) are indicated. Both deletion strains show enhanced ampicillin sensitivity over wild-type.
(B) Relative optical densities 6 hr after inoculation for WT (black), yajc (blue), and acrb (green) E. coli deletion strains in the presence of nafcillin. Nafcillin concentrations (0 g/l, 0.01 g/l, 0.05 g/l, 0.25 g/l, 1 g/l, 4 g/l) are indicated. Only the acrb deletion strain shows significantly enhanced nafcillin sensitivity over wild-type.
Error bars represent the maximum deviation from the average.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions

7. Figure 6 Schematic Representation of the Complete Efflux Pump
AcrB (orange), TolC (gray), and AcrA (blue) in complex with YajC (green). A rotation (orange arrow) observed for the porter domain of AcrB due to interactions with YajC is suggested to be communicated to TolC (gray arrow) by interactions with AcrA and to thereby open the periplasmic entrance to the pore.
Structure  , DOI: ( /j.str )
Copyright © 2007 Elsevier Ltd Terms and Conditions