(A) A 1 µs MD snapshot of the pre-protein from the SecA-SecYE-PP complex with ATP bound, overlaid on the SecA-SecY schematic from Figure 1C. A region of α-helix, as computed by the dictionary of secondary structure of proteins (DSSP) and confirmed visually, is visible in the exterior cavity. The approximate position of the membrane is shown, and each nine residue stretch of pre-protein used for folding analyses, respectively in the exterior and cytoplasmic cavities, are indicated by dashed lines. (B) Deformation analysis of the pre-protein within SecA-SecYEG. SecA-SecY is shown as a schematic, and the pre-protein is shown as tube, color-coded and sized according to its deformation energy (low deformation blue and thin; high deformation red and wide). Analysis reveals three major sites with high deformation energy; at the beginning of the signal sequence, in the SecY cytoplasmic cavity, and in the SecA ring. The former is more pronounced in the ATP state, the latter two in the ADP-bound state (see Figure 2—figure supplement 4A). (C) The degree of pre-protein folding in the exterior and cytoplasmic cavities of SecY in the ADP and ATP states. Shown are the combined datasets for the mirrored and tandem substrates (see Materials and methods for details), in either a simple or more complex bilayer supplemented with negative phospholipids (PG and cardiolipin). Data are collected from the ATP (blue) or ADP (red) bound states. There is a marked difference in degree of asymmetry in the ATP data, but not the ADP (p<0.0001 and p=0.0690 respectively, from two tailed t-tests). Error bars are s.e.m. The breakdown of data between uniform bilayers and those supplemented with negative phospholipids can be seen in Figure 2—figure supplement 4E. (D) As panel C, but showing the degree of folding in bulk water; that is, not in the presence of translocon. These analyses used the same pre-protein starting coodinates as panel C. In both the ATP (light blue) and ADP (pink) data, there is no significant difference between the cytoplasmic and exterior regions of pre-protein. Reported p values are 0.15 and 0.98. Error bars are s.e.m. (E) Comparison of folding data set with the wild-type signal sequence (blue; as per panel C) and a defective signal sequence (SSΔ4; green). The introduction of a defective signal sequence significantly increases the degree of pre-protein secondary structure in both the cytoplasmic and exterior cavities (p=0.021 and p=0.002). The breakdown of data can be seen in Figure 2—figure supplement 4B.