Extended Data Fig. 7: MD simulations to examine putative lipid translocation pathways through LetA, related to Fig. 4.

a, b, Cartoon representation of uncrosslinked (a) or crosslinked (b) LetA (gray) with EM density (orange) corresponding to Lipid 1 (yellow; Map 2a for uncrosslinked LetA and Map 1a for crosslinked LetA). Figures were prepared in ChimeraX¹¹² using the zone function with an applied radius of 3.5 Å and map contour of 0.124 for Map 2a or radius of 3.5 Å and map contour of 0.00539 for Map 1a. Gray spheres indicate the residues that are interacting with Lipid 1. Nitrogen and oxygen atoms are highlighted in blue and red, respectively. Residues replaced with BPA in crosslinking experiments presented in (b) are shown as pink spheres (W162 and W267). W267 is exposed to bulk lipids and serves a positive control. W127 is predicted to interact with one of the Lipid 1 tails. c, SDS-PAGE analysis of purified LetAB and its BPA mutants, either crosslinked or uncrosslinked and stained by Coomassie (LetB) or phosphor-imaged (³²P signal). Three replicates of the experiment were performed starting with three different colonies, on different days. d, Diagram showing the MD simulations performed and the movies associated with each step. e, Time series of the z-position of the phosphorus atom of Lipid 1 in each replica. Lipid 1 was either included (+Lipid 1) or excluded (−Lipid 1) at the start of the simulation. Except in Replica 2 (−Lipid 1), a phospholipid binds to the Lipid 1 binding site regardless of whether a lipid was modeled in it to begin with or not (there is no trace for Replica 2 (−Lipid 1) in the right panel, since no lipid is stably bound). The starting z-positions of the phosphorus atom are marked by circles (see color key). The average phosphorous atom position from the lipids within 10 Å of LetA is shown as a black line. f, Representative snapshots of Lipid 1 in Replica 1 (+Lipid 1) of the equilibrium MD simulation, showing its stable binding throughout the simulation. Each Lipid 1 snapshot is colored according to the simulation timestep, transitioning from light yellow at the start to orange at the end. LetA is shown in blue, MCE Ring 1 in purple, and phosphorus atoms of the bulk lipids are in white spheres to indicate the position of the membrane. Helices are depicted as cylinders. g, Time series of the z-position of the phosphorus atom from the most elevated lipid in the central cavity. The lipid type is indicated in the color key. The starting z-position of the phosphorus atoms are indicated by a circle. The average phosphorus atom position of lipids within 10 Å of LetA is depicted as a black line. h, Accumulated non-equilibrium work profiles for different pulling protocols and the pulled lipid types (PMPE and PYPG). The first and second circles on each line indicate the time when the pulled lipid reached the bottom and middle of the periplasmic pocket, respectively. At the end of each line, the lipid has reached the top of the periplasmic pocket. i, RMSD profiles of steered lipids. RMSD plots for each lipid type (PMPE and PYPG) and different pulling methods (head group, one tail, or two tails) showing lipid movement and flexibility within the periplasmic pocket during the 300-ns equilibrium simulations after SMD. j, The conformational dynamics of the steered lipids are illustrated in 2D scatter plots showing the orientation angle versus the z-position of the phosphate group. Time progression is indicated by a color gradient from dark blue to yellow, with the initial and final frame highlighted by a circle (see color key). The orientation angle, as shown in the schematic on the right, is defined by the angle between the orientation vector (yellow) and the membrane normal (black). k, Western blot analysis of cell lysates of the strains indicated, to compare cellular levels of WT LetB and LetB R63 mutants. LetB was probed using a α-LetB antibody. As a loading control, BamA levels were detected using an α-BamA antibody. WT LetA was co-expressed with each LetB mutant. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. l, Sequence logo of positions 58 to 68 of LetB by WebLogo 3 (see Methods for list of LetB sequences). m, The fold change between purified LetAB and the membrane fraction. The black dotted line indicates no change in the indicated lipid class. Error bars indicate mean ± standard error of the mean from three independent LetAB purifications and three technical replicates of each (n = 9), each shown with a dot. P-values were calculated from standardized z-scores propagated through the fold change calculation. The Benjamini-Hochberg method was used to correct for multiple comparisons. The corrected p-values for CL, PE, and PG were <10⁻¹², 2.2 × 10⁻⁶, and 0.014, respectively (*** p < 0.001). n, The relative abundance of phospholipids from purified LetAB versus the membrane fraction of E. coli cells overexpressing LetAB. The normalized summed intensity was used to estimate abundance. Error bars indicate mean ± standard error of the mean from three independent LetAB purifications and three technical replicates (n = 9), each noted with a dot. A two-tailed t-test with a 95% confidence interval was used to compare the normalized means of each lipid class area between the protein and membrane samples. The Benjamini-Hochberg method was used to correct for multiple comparisons. The corrected p-values for CL, PE, and PG were 1.17 × 10⁻⁶, 4.15 × 10⁻⁵, and 0.059, respectively (*** p < 0.001). Gel source data for panels (c) and (k) are provided in Supplementary Fig. 1m,n, respectively.