Extended Data Fig. 9. Examples of mutations clustering at intermolecular interaction sites, whose location suggests novel mechanisms of HCM pathogenicity (zoom for detail).

The map reveals that many HCM pathogenic mutations in cMyBP-C, and in myosin tails and heads of specific crowns (CrH, CrT and CrD), are in intermolecular interfaces in the native filament. These mutations could not be mapped previously due to use of the tarantula filament IHM, lacking cMyBP-C and titin^5,6. a, Mutations affecting the tail-titin interface. Mutations in ring 1 (D906G) and 2 (E924K, E930K) of CrD S2⁴⁸ occur at the site of interaction with TB domains T5 and T6. Surface charge depiction showing positively charged patches on TB (blue on right inset) suggests that these mutations, involving loss or reversal of negative charge, would weaken binding of CrD S2 to TB. This could interfere with transmission of tension by titin to the cMyBP-C–TaD binding site, and thus disrupt possible mechanical signaling mechanisms (see text and ED Fig. 10). b, Mutations affecting tail-tail interfaces. Mutation E924K in (a) is also involved in a tail-tail interaction, in this case not CrD, but the S2s of CrH and CrT. E924K charge reversal on CrT S2 (right inset) would be expected to weaken interaction with positive charge (blue) on CrH S2, thus impairing stability of the tail network. Disruption of function in these 2 different ways (tail-TB and tail-tail) could make the E924K mutation especially pathogenic. c, Mutations in cMyBP-C and the myosin motor domain affecting the cMyBP-C–myosin head interface. Pathogenic mutations in the myosin head (P307H) and the cMyBP-C C8 domain (R1002Q, E1017K) both occur in the interaction interface of C8 with the FH motor domain of CrH. Surface charge depiction suggests that charge loss or reversal on C8 (left inset) and on CrH FH (right inset) would weaken this interface, impairing cMyBP-C’s stabilization of the CrH FH. This could disturb its SRX state and interfere with proposed mechanical signaling mechanisms (ED Fig. 10). d, Mutations in the myosin motor domain affecting multiple interfaces with tails. The classic mutation R403Q, in the CM loop of the motor domain, has been widely studied but not fully explained⁷³. The reconstruction suggests that it could have multiple effects, by impairing head-tail interactions differently in CrH and CrT FHs. In the upper inset, the CrT CM loop interacts with its own S2, while in the lower, the CrH CM loop interacts with S2 from a more distal CrT. Loss of positive charge in the former may strengthen binding to the positive charge K939, stabilizing the IHM, while in the latter it would weaken interaction with the CrT tail (E1119/E1120), again affecting mechanical signaling mechanisms (ED Fig. 10). In summary, due to the various environments of myosins in a single 430-Å repeat, a single mutation can affect multiple interactions, with different pathogenic consequences.