Structure of the mitochondrial ATP synthase by electron cryomicroscopy

Abstract

We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 A resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic alpha-beta interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis.

Fig. 1. EM of ATP synthase particles. Frozen ATP synthase particles were imaged with a 200 kV electron microscope. (A) A sample region from a micrograph. (B) Some typical particle images. The scale bar in (A) represents 200 Å and the scale bar in (B) represents 100 Å.

Fig. 2. Class-average images of the ATP synthase. (A) Average of all of the aligned images in the dataset. The scale bar represents 100 Å. (B–E) Class-averages that probably arise from incoherently averaged particle images (and consequently, like the overall average, have a line of symmetry). (F–M) Class-averages where a corresponding mirror image is found (e.g. F with J, G with K, etc.) and constitute views about the long axis of the complex. (N and O) A mirror pair, but each probably consisting of averages of two non-equivalent views of the assembly with the long axis tilted from the plane of the grid. (P–U) Class averages that have no mirror pair in the dataset.

Fig. 3. Rotation analysis. (A) Features that deviated from cylindrical symmetry in class-averages of Figure 2F–M were emphasized by subtracting the average of all of the views from each of the views. Markers were placed at maxima in the density to indicate the corresponding asymmetric features in each image. (B) To compare the experimental marker positions to the hypothesis that the images of the ATP synthase constitute a single-axis rotation series, the ratio of marker position to calculated marker radius (a_i,j/r_i) was plotted against the total angle for each marker in each image (φ_j + δ_i). The observations are consistent with the hypothesis that the series constitutes a rotation sequence, indicated by the dashed line (cos[φ_j + δ_i]).

Fig. 4. 3-D model of the ATP synthase. (A and B) Surface rendered views of the model after refinement. (C) The model after being divided into two parts. The first (blue) was chosen to correspond to the F₁-c₁₀ subcomplex and the remaining density (green) is interpreted to represent the peripheral stalk and second domain of F_O. The grey mesh represents the experimental EM map.

Fig. 5. Two further views of the peripheral stalk. (A) A side-view of the model shows the curvature of the peripheral stalk. (B) A view from the F₁ end of the assembly along its long axis reveals a sharp bend in the peripheral stalk near to its terminus.

Fig. 6. Determination of the absolute hand of the model. Tilt pairs of 29 different particles were recorded with a tilt between images of 30°. The first particle in each pair was aligned to the model. The plot shows the average phase residual between the model and the second particle in each pair for all possible tilt axes and tilts up to 40°. From the known direction and magnitude of the tilt between the images in the pair, a model of the correct hand was expected to produce a minimum at (2,–30°) as opposed to (–2,30°). The observed tilt axis and amount of tilt indicate that the model, as shown, is correct.

Fig. 7. Docking of an atomic model of F₁-c₁₀ into the EM map. The volume of the EM map not occupied by the atomic structure is coloured green. (A) A side-view of the model. The density that cannot be explained by the F₁-c₁₀ model consists of the peripheral stalk and the second domain of the F_O region. This density must contain the remaining subunits of the ATP synthase: a, b, d, e, f, g, A6L, F₆ and OSCP. (B–D) Cross-sections of the model. The terminus of the peripheral stalk at the top of F₁ is on the central axis at the apex of the model (B). The peripheral stalk is primarily in contact with an α-subunit (red) near the top of F₁, but is in contact with a non-catalytic interface between an α- (red) and β- (yellow) subunit along most of F₁. The matching flattened sides of the F₁ in the crystal structure and EM map show the uniqueness of fit between the two models. The central stalk and c-ring fit into the model well (D).