Extended Data Fig. 6. Different conformations of titins TB and TA (zoom for detail).

a, The two titin densities (TB, TA) in one 430-Å super-repeat of a sector contain 11 Ig and Fn domains, and their overall conformation is not straight. The titin maps segmented from the reconstruction show that, due to the presence of 3 kinks (arrows), and a slight curve in the T1–T3 and T8–T11 regions, one super-repeat (11 domains) fits precisely into 430 Å, matching exactly the triplet repeat of myosin heads (CrD-CrT-CrH). The kinks, all occurring at an Ig-Fn junction, demarcate three parts of the super-repeat: 1. T1–T3 (Ig-Fn-Fn); 2. T4–T7 (Ig-Fn-Fn-Fn); and 3. T8–T11 (Ig-Fn-Fn-Fn). Parts 1 and 3 of TB and TA have similar conformations, and are approximately parallel to each other. However, the conformation of Part 2 differs between TB and TA. In TB, T4–T7 is rotated ~90° with respect to TA, positioning it at a higher radius above the surface of the backbone, where it becomes available for interaction with CrD S2 as shown in b (see also Supplementary Video 1). b, Because of this conformational difference, the middle portions of TB and TA interact differently with different tails. The bent and raised conformation of T5–T6–T7 in TB binds to proximal S2 of CrD with mostly electrostatic attraction (inset), while the straight conformation of T5–T6 in TA binds to distal S2 of CrH (ED Fig. 7), generating an axial shift of 148 Å between CrH and CrD (see d). c, Previous studies speculated that the 39 Å reflection in the X-ray diffraction pattern of relaxed muscle (ED Fig. 3g) may be due to titin⁷². Here we demonstrate this directly. Fast Fourier transformation (FFT) of the titin strand, segmented from the reconstruction in its native (kinked) conformation (right), produces a meridional reflection at a spacing of 39 Å, the 11^th order of the 430 Å repeat (seen as layer lines at lower angles). If titin is computationally straightened (left), the reflection moves towards the origin (~44 Å spacing), producing a reflection that is not observed in the relaxed X-ray pattern. Agreement of the reconstruction with X-ray data from relaxed intact muscle (ED Fig. 3g vs. e, f) implies that titin is kinked in the native state, allowing the 11 domains of its super-repeat to fit into 430 Å. The FFTs were computed from 8 titin super-repeats of the kind shown in c, laid end-to-end, to create a strand similar to that in one C-zone of the thick filament. See also ED Fig. 10. d, Past studies suggested that the uneven spacing of titin’s 3 Ig domains in the super-repeat might be responsible for the uneven spacing of the 3 myosin crowns in the 430 Å repeat. Our reconstruction enables us to test this idea. We find an approximate correlation between the positions of the 3 Ig domains (purple) and the motor domains of the 3 IHMs. Ig1 (T1) correlates with CrT, Ig2 (T4) with CrD, and Ig3 (T8) with CrH. But we see no precise correlation that would suggest that the 3 Ig domains directly position the crowns (see crown and Ig spacings on figure). This is not surprising, given the absence of any direct titin-head interaction in the structure. Instead, titin positions the crowns through interaction of TB and TA with CrH and CrD tails, while CrT is positioned by interaction of TaT with TaH (see text). See also Supplementary Video 5.