Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

Abstract

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

Keywords: ATP synthase; biochemistry; biophysics; bovine; coevolution; cryo-EM; evolutionary covariance; structural biology; structure.

Figure 1.. Cross-sections through maps.

(A) Cross-sections through the F₁ regions of the different maps show that states 1, 2, and 3 are related by ∼120° rotations of the γ subunit within the α₃β₃ hexamer (blue arrows). (B) Surface rendering of a map (State 1a) shows the bent F_O region with a tubular feature that extends from the rotor-distal portion to the c₈-ring (orange arrow). (C) Cross-sections through the F_O region show α-helices from the a, b and A6L subunits (green arrows), a low density region in the rotor-distal portion (white arrow), the tubular extension from the rotor-distal portion to the rotor (orange arrows), and the c-ring (purple arrows). (D) Averaging the F_O regions from the seven different maps shows all of the features mentioned above with an improved signal-to-noise ratio for some features. Scale bars, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.003

Figure 1—figure supplement 1.. Electron microscopy and model construction.

(A) A sample micrograph with representative ATP synthase particles circled in blue. Scale bar, 500 Å. (B) A sample micrograph power spectrum with the modeled power spectrum from CTFFIND4 in the lower left quadrant. (C) Representative averages from 2D classes selected for 3D analysis. (D) An example of individual particle trajectories (exaggerated by a factor of 5) from alignparts_lmbfgs used for local drift correction. (E) The distribution of Euler angles assigned to particles going into the 3D maps shows preferred orientations but contains sufficient particle views to produce maps with isotropic resolution. DOI: http://dx.doi.org/10.7554/eLife.10180.004

Figure 1—figure supplement 2.. The seven observed states of the bovine mitochondrial ATP synthase.

Two views are shown for each of the seven conformations identified for the enzyme. All of the known structural features and the newly observed protuberance from the rotor-distal portion of the F_O region are seen in each map. The bend in the F_O region is indicated by the dashed line in the top left map. Scale bar, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.005

Figure 1—figure supplement 3.. Fourier shell correlation curves for the seven maps.

FSC curves are shown for state 1a and 1b (A), state 2a, 2b, and 2c (B), and state 3a and 3b (C). DOI: http://dx.doi.org/10.7554/eLife.10180.006

Figure 2.. 3D structure of the F_O region.

(A) In the F_O region of the complex, density was segmented for the a subunit (green), the b subunit (red), the A6L subunit (blue), and the structure thought to arise from subunits e and g (orange). (B) The a subunit sequence could be placed unambiguously into the cryo-EM density (green) by including constraints for residues predicted to be near to each other due to evolutionary covariance (red lines). (C) The a subunit (coloured with a gradient from blue to red to denote directionality from the N to C terminus) possesses six α-helices, numbered 1–6. Trans-membrane α-helices from subunits b and A6L are shown as volumes (red and blue, respectively). Five of the α-helices of subunit a are membrane-inserted while helix #2 runs along the matrix surface of the F_O region. The N terminus of the a subunit is on the inter-membrane space side of the subunit while the C terminus is on the matrix side. The highly conserved residue Arg159 is on the elongated and highly tilted α-helix #5. Scale bar, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.008

Figure 2—figure supplement 1.. Analysis of evolutionary covariance of residues.

(A) The top 90 predicted couplings between residues of the a subunit are indicated, along with trans-membrane helices predicted by the MEMSAT-SVM algorithm (52), shown in green, and highly conserved residues, shown in red. Residues modeled as membrane-inserted α-helices based on the cryo-EM density are indicated with dark blue rectangles outside of the sequence and residues modeled as a soluble α-helix based on the cryo-EM density is indicated with a light blue rectangle. (B) The top six predicted couplings between residues of the a subunit and residues on the outer surface of the c-ring are indicated. DOI: http://dx.doi.org/10.7554/eLife.10180.009

Figure 3.. Docking of atomic models into the cryo-EM maps.

Fitting of all available atomic models into the density map is shown for state 1a (A) and state 1b (B). State 1a is also shown in a different orientation and without the density map (C) and with the c₈-ring removed for clarity (D). The apparent gap between the c₈-ring and γ and δ subunits is filled with amino acid side chains and is the same as was seen in the crystal structure of the F₁-c₈ complex (Watt et al., 2010). Scale bar, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.011

Figure 3—figure supplement 1.. Differences between sub-states.

The differences between sub-states can be seen by overlaying maps and models for state 1a (red) and 1b (green) (A), 2a (red) and 2b (green) (B), 2b (red) and 2c (green) (C), and 3a (red) and 3b (green) (D). These differences can be represented approximately as a rigid body rotation of the ₃₃ hexamer by 10 (state 1a to 1b), 11 (state 2a to 2b), 12 (state 2b to 1c), and 16 (state 3a to 3b). The axes of these rotations are shown as black rods. This movement is most easily seen in Videos 4 and 5. Scale bar, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.012

Figure 4.. Model for proton translocation.

(A and B) The a subunit (green), along with the membrane-intrinsic α-helices of the b subunit (red), form two clusters that could be the half channels needed for trans-membrane proton translocation. (C and D) The map segment corresponding to the c₈-ring is shown for state 2a (pink) and state 2c (purple). The difference in rotational position of the c-ring is consistent with the Brownian fluctuations predicted for the generation of a net rotation. Scale bar, 25 Å. DOI: http://dx.doi.org/10.7554/eLife.10180.016