Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs

Fig. 1. Electron microscopy of epon-embedded cardiac myofibrils. a, b Slices of tomograms showing the cross-sections of the Z-discs in the EGTA + ATP and the Ca + ATP states. Black and white of the images were inverted. c, d Slices of averaged subtomograms showing the actin lattices. The square lattice of the EGTA + ATP state and the diamond-shaped lattice of the Ca + ATP state were shown.

Fig. 2. Cryo-electron microscopy of native cardiac myofibrils. a Low-magnification image of a thin myofibril in the EGTA + ATP state. Broken box indicates the recording area in b. b 0° tilt image of the Z-disc. c Slice view of the reconstructed tomogram. Red broken line indicates the position of the cross-section in d. d Cross-section of the tomogram of the Z-disc. e Lattice points visualized by enhancing the contrast of the cross-sectional view. These lattice points were manually picked for extraction of the subtomograms.

Fig. 3. Subtomogram averaging of the repeat unit of the Z-disc. a, b Averaged subtomograms composed of the central F-actin (gray), the opposite-polarity F-actins (yellow), and the α-actinin molecules (orange). Eye symbols on the top views indicate the direction of the side views on the right. (+): barbed end; (−): pointed end. Top views are seen from the pointed end of the central F-actin. c, d Combination of the six shifted maps with the original map. Coordinates were re-centered to the neighboring six F-actins (four opposite polarity F-actins, and two parallel F-actins) and additional 3D refinements were conducted based on the new coordinates. The resulting shifted maps were then superimposed by aligning one F-actin of the shifted map with the corresponding F-actin of the original map, as indicated by the red arrows. e, f Composite maps created by combining seven maps. The EGTA + ATP and the Ca + ATP states show the basket-weave and the diamond-shaped lattice conformations, respectively. Both monomers of the α-actinin dimer (orange and green) were fully visualized in the combined maps. Eye symbols on the top views indicate the direction of the side views on the right.

Fig. 4. Multibody refinement of the F-actin and the α-actinin. a Composite maps composed of one central F-actin (gray), four opposite-polarity F-actins (yellow), sixteen α-actinin monomers (orange and green). The α-actinin crystal structures (PDB 4D1E) were fitted into the α-actinin maps (red and green models). Eye symbols indicate the direction of the side views on the right of the respective top views. (+): barbed end; (−): pointed end. b Visualization of the first and the second eigenvectors (Supplementary Fig. 1d). In both states, the first eigenvector represents tilting of the central F-actin about the center of the filament, and the second eigenvector represents rotation of the opposite-polarity F-actin about the central F-actin.

Fig. 5. Conformational changes in the α-actinin. a Top view of the actin-actinin cross-bridging complex. Gray: central F-actin; yellow: opposite-polarity F-actin; orange and green: α-actinin monomers. The maps of the EGTA + ATP and the Ca + ATP states are light- and deep-colored, respectively. Eye symbols indicate the direction of the side views in b, c. b The α-actinin dimer twisted about its long axis and the opposite-polarity F-actin showed a ~13° turn. The α-actinin dimer showed a ~21° swing, which translocates the opposite-polarity F-actin by ~80 Å. (c, insets) Close-up views of the interface between the central F-actin and the actin-binding domain of the α-actinin. Eye symbols indicate the direction of the close-up views. The F-actin models (PDB 6KLL) were fitted into the central F-actin maps.

Fig. 6. Angular mismatches between the helical and the lattice symmetry. The rotation angles of the F-actin helix and the square/diamond-shaped lattice of the Z-disc were compared. The α-actinin molecules (transparent red) regularly bind to the central F-actin. The α-actinin-bound actin monomers are colored red or blue. The one-start helix of the F-actin with 27.5 Å-rise and 166.6°-twist can be regarded as a two-start helix with 55 Å-rise and –26.8°-twist. The “helical angles” are the rotation angles calculated from the helical symmetry of the F-actin relative to one of the actin monomers designated as “0°”. The “lattice angles” correspond to the rotation angles of the opposite polarity F-actins around the central F-actin. In the EGTA + ATP state, the lattice angles are an arithmetic progression with common difference of 90°. In the Ca + ATP state, by contrast, the rotation between the two-neighboring opposite-polarity F-actins is either 80° or 100°. Although the square lattice favors the one-start helix model because of the nearly 90°-turn per seven monomers (asterisks), there are discrepancies in other positions (e.g. 90° versus 107.2°). On the other hand, the diamond-shaped lattice…