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. 2020 Dec 15;9:e62163. doi: 10.7554/eLife.62163

Figure 5. The ATP-dependent transport cycle of Dnf1–Lem3 and Dnf2–Lem3.

(A) The five structures of Dnf1 and Dnf2 in the E1 (apo), E1-ATP (AMPPCP), E1P-ADP (AlF4−-ADP), E2P-transition (AlF4−), and E2P (BeF3−) states. The A domain in the E1, E1-ATP, and E1P-ADP states is flexible and is shown here as a ribbon in the yellow rectangle to indicate its position. The structures of the E1-ATP and E1P-ADP states within the black dashed box are almost identical. The structures of the E2P and E2P-transition states are similarly grouped for the same reason. Magenta arrows indicate the domain movement during the Post–Albers cycle in the order E1 to E1-ATP to E1P-ADP to E2P/E2P-transition. (B) Structural comparison of the substrate-binding sites in the E1, E1P-ADP, and E2P states. TMH1-2 in the E1 and E1P-ADP states tilt with respect to that in the E2P state; this movement closes the PL1 site and opens the PL2 site.

Figure 5.

Figure 5—figure supplement 1. High structural similarity between Dnf1 and Dnf2 in the corresponding substrate-transport states.

Figure 5—figure supplement 1.

(A) Structural alignments 3D maps of Dnf2–Lem3 in the E1-ATP and E1P-ADP states. (B) Structural alignments of 3D maps of Dnf2 in the E2P and E2P-transition states.
Figure 5—figure supplement 2. The N and A domain densities are weak in the 3D maps of Dnf1–Lem3 in the E1 and E1P-ADP states, indicating their high flexibility.

Figure 5—figure supplement 2.

(A) Cryo-EM 3D map of the Dnf1-Lem3 in the E1 state displayed at a high (top panel) and a low threshold (bottom panel). Red arrows indicate the domain movement from the E1 state to the E1-ATP state. (B) Cryo-EM 3D map of the Dnf1–Lem3 in the E1-ATP state displayed at a high (top panel) and a low threshold (bottom panel). Red arrows indicate the domain movement from the E1P-ADP state to the E2P state. (C) Cryo-EM 3D map of the Dnf1–Lem3 in the E2P state. (D–F), Selected reference-free 2D class averages of the Dnf1–Lem3 in the E1 state (D), in the E1P-ADP state (E), and in the E2P state (F). The averages in red or green boxes belong in the same views. The A domain locations are labeled by red arrowheads. The weak and blurry A domain density indicates its flexibility in the E1 and E1P-ADP states.