Comparing cryo-EM structures of the vertebrate cardiac muscle thick filament

Fig. 1. Myosin and thick filament structure. (a) Myosin 2 molecule showing tail, heads, and light chains. (b) Basic structure of bipolar thick filament, with tails in backbone, heads on surface, and central bare zone. (c) Vertebrate filament showing ordering of heads with a 430 Å repeat, and associated proteins MyBP-C, defining the C-zone (C), and titin. Red spheres represent pairs of myosin heads in quasi-helices (yellow lines), green bars show 430-Å spaced positions of MyBP-C, backbone is cyan, titin (pink) runs along each half thick filament and has spring-like properties beyond the filament tip, in the I-band. Z-line, M-line, P zone and D zone are also marked. (d) PDB 1i84: Interacting heads (blocked and free), first identified by cryo-EM of 2D crystals of smooth muscle heavy meromyosin (Wendt et al. 2001). BH and FH heavy chains are green and dark blue; light chains are in narrow neck region of each head. S2 has been omitted from the PDB as its position was unknown. (e) Cryo-EM reconstruction of tarantula thick filament (Woodhead et al. 2005) showing repeating J-shaped motif in 4 helices (two on front arrowed), with 430 Å repeat and 143 Å between crowns. (f) Fitting of myosin head…

Fig. 2. Averaged 3D maps of C-zone structure from the 3 cryo-EM studies. (a) Reconstruction of 5 crowns of IHMs in human cardiac thick filament, based on single particle analysis of isolated filaments in presence of mavacamten (Dutta et al. 2023). The structure repeats every 3 crowns (430 Å). Using the naming scheme of (Dutta et al. 2023), Crown T (CrT) has tilted IHMs, crown H (CrH) has horizontal IHMs, crown D (CrD) has (partially) disordered IHMs (wispy surface). The labels below CrT, CrH and CrD show the naming schemes for the equivalent crowns from the reconstructions in (Tamborrini et al. 2023) (b) and (Chen et al. 2024) (c), respectively. (b) Reconstruction of 430 Å repeat of C-zone (3 crowns) of mouse cardiac thick filament based on cryo-ET of relaxed myofibrils in presence of mavacamten (Tamborrini et al. 2023). Crowns 1, 2 and 3 correspond to crowns CrH, CrD and CrT (see (a)). Titin is pink, MyBP-C is yellow. (c) Reconstruction of 5 crowns of human cardiac thick filament based on cryo-ET of frozen human ventricle fragments in absence of mavacamten (Chen et al. 2024). Crowns IHM-D, IHM-S and IHM-C correspond to crowns CrD, CrT and CrH in (a). Reconstructions shown to scale and…

Fig. 3. 3D reconstructions of the C-zone from the 3 studies showing organization of MyBP-C domains. (a) In isolated thick filament (Dutta et al. 2023), MyBP-C (pink) runs longitudinally along the surface of the filament from C10-C2. C10 and C9 are hidden behind the CrT IHM. Titin strands orange, yellow. (b) In intact myofibril (Tamborrini et al. 2023), with thin filaments surrounding thick filaments, the C-terminal domains C10-C8 run longitudinally, while the molecule pivots at C7 to extend transversely, at varying angles, towards the thin filament. MyBP-C yellow, tails blue, titin pink. (c) In intact myofibril in absence of mavacamten (Chen et al. 2024), the organization of MyBP-C domains is similar to that in its presence (b): C10-C8 run longitudinally, while at C7 the molecule pivots to run transversely. (a) from (Dutta et al. 2023); (b) modified from (Tamborrini et al. 2023); (c) from (Chen et al. 2024), reproduced under Creative Commons license https://creativecommons.org/licenses/by-nc-nd/4.0/)

Fig. 4. CrD position is different in isolated filament compared with filament in myofibrillar lattice. The atomic model from the isolated filament reconstruction PDB 8g4l (Dutta et al. 2023) is fitted into the translucent cryo-ET map of the C-zone of the thick filament in the filament lattice (EMD-18146; (Tamborrini et al. 2023)). The fit demonstrates the excellent agreement between the two structures in the positions of the CrH and CrT IHMs, the tails (red), titin (TA and TB, purple), and of MyBP-C (grey) from domain C10 to C8, after which the two diverge. C7 projects away from the filament in the cryo-ET map while domains C7-C5 continue along the surface of the isolated filament reconstruction. Viewing in three dimensions is required to fully appreciate this difference (Movie S1). The structures are strikingly different for CrD, which is displaced ~ 70 Å azimuthally between the two (double arrowhead). This suggests a substantial influence of neighboring thin filaments on CrD position