Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

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1 Supplementary Figure 1 Chemical structure of LPS and LPS biogenesis in Gram-negative bacteria. a. Chemical structure of LPS. LPS molecule consists of Lipid A, core oligosaccharide and O-antigen. The polar part of Lipid A is negatively-changed due to the presence of two phosphate groups. b. Ra-LPS molecule is approximately 32Å in height and 28 Å 12 Å in the other two dimensions. The dimensions of an Ra-LPS is based on the crystal structure of TLR4/MD-2/Ra-LPS complex (PDB ID: 3FXI). c. LPS biogenesis in Gram-negative bacteria. After flipped to the IM outer leaflet by MsbA, LPS is extracted from IM, transported cross the periplasm and finally inserted in the OM by the LptABCDEFG transenvelope complex. LptB 2FG is a quaternary ABC transporter.

2 Supplementary Figure 2 The electron density maps of LptB 2FG. a. Stereo views (cross-eyed) of the 2F o-f c electron density map for the complete LptB2FG complex structure at 3.46Å. b. The 2F o-f c electron density maps of representative regions of the TMDs of LptFG (TM1-LptF and TM1-LptG) are shown. Selenomethionine residues (in red) and bulky residues are used as makers for guiding model building. c-d. Validation of side-chain register of the nucleotide-free LptB 2FG transporter. Anomalous electron density maps define selenomethionine (contour level: 3.0σ) in (c) and Pt sites (contour level: 4.5σ) in (d). In (c), anomalous density was observed for 28 out of 32 selenomethionines of the complete LptB 2FG complex.

3 Supplementary Figure 3 Domain organization of the LptB 2FG complex. a. Domain organization of the LptB 2FG complex. The two ATPase domains (LptB) in cytoplasm are colored in cyan and green. The TMD domains of LptF and LptG, each containing six transmembrane helices, are colored in violet and yellow, respectively. The two periplasmic β-jellyroll domains of LptF and LptG that stem from TM3 and TM4 of LptF(G) are colored in grey. The two coupling helices of LptF and LptG connecting TM2 and TM3 of LptF(G) in cytoplasm are highlighted in blue. b. Overlay of the TMD of LptF with that of LptG. LptF and LptG are colored in violet and yellow, respectively.

4 Supplementary Figure 4 Sequence alignment of LptF homologs and residues selected for functional analysis in the structure. a. Sequence alignment of LptF homologs from five representative Gram-negative bacterial strains. b. Conserved hydrophobic and positive residues of LptF lining the V -shaped cavity selected for mutational studies. Conservation of LptF residues in different Gramnegative homologs is shown in ENDscript. Secondary structures are numbered within the respective domains. Conserved residues lining the inner surface of the V -shaped cavity were selected for mutational analyses are highlighted. The labeled residue types and numbers in both alignments correspond to those in E. coli.

5 Supplementary Figure 5 Sequence alignment of LptG homologs and residues selected for functional analysis in the structure. a. Sequence alignment of LptG homologs from five representative Gram-negative bacterial strains. b. Conserved hydrophobic and positive residues of LptG lining the V -shaped cavity selected for mutational studies. Conservation of LptG residues in different Gramnegative homologs is shown in ENDscript. Secondary structures are numbered within the respective domains. Conserved residues lining the inner surface of the V -shaped cavity were selected for mutational analyses are highlighted. The labeled residue types and numbers in both alignments correspond to those in E. coli.

6 Supplementary Figure 6 Mutagenesis study of the conserved residues that line the inner surface of the V -shaped cavity in the TMDs of LptF and LptG. The growth phenotypes of the lptfg-depleted E. coli strain NR1113 transformed with various hydrophobic-to-hydrophilic LptG_Ec mutants (a) and LptF_Ec mutants (b) on LB agar plates containing 0.1% L-arabinose and 50 μg ml 1 kanamycin. The growth phenotypes of the lptfg-depleted E. coli strain NR1113 transformed with positive-to-negative mutations in the absence of L-aribinose (c), mutant protein expression levels (d) and the growth phenotypes in the presence of 0.1% L-arabinose (e). Mutational analyses of residues from the coupling helices of LptF_Ec and LptG_Ec on LB agar plates containing 0.1% L-arabinose and 50 μg ml 1 kanamycin (f). All labeled residue types and numbers correspond to those of LptF_Ec and LptG_Ec. In the presence of 0.1% L-arabinose, the

7 lptfg-depleted E. coli strain NR1113 transformed with WT, vector control and various mutants all grew well similar to that of WT. In the absence of L-arabinose, the lptfg-depleted E. coli strain NR1113 transformed with pqlink-kan vector and plasmids encoding wild-type (WT) LptFG were used as negative control and positive control, respectively. All the complementation assays were repeated at least three times and a representative result is shown.

8 Supplementary Figure 7 Three representative ABC exporters in their inward-facing or outward-facing conformational states. The nucleotide-free LptB 2FG transporter (a), the heterodimeric TM287-TM288 exporter in the apo state (PDB code: 4Q4H) (b) and the heterodimeric nucleotide-free human sterol ABCG5-ABCG8 exporter (PDB code: 5DO7) (c) in inward-facing conformational state; the homodimeric AMP-PNP-bound Sav1866 exporter (PDB code: 2ONJ) (d) in outward-facing conformational state.