Figure 3. A unique contact is established upon transition from ADP to rigor.

(A) View of the LPF APD MDFF model (dark magenta) and LPF rigor MDFF model superimposed in the reference frame of the actin filament (light gray density). To generate this superposition, the ADP and rigor density maps were aligned, then their corresponding atomistic models were rigid body fit into the aligned maps. (B) Minimal actin binding cleft rearrangements are observed between ADP and rigor, superimposed as described in A. ADP U50, magenta; ADP L50, dark magenta; rigor U50, dark grey; rigor L50, light grey; actin density, white. Arrows denote displacements of domain centroids (spheres) from ADP to the rigor state. Centroids were determined for U50 (residues 180–206, 229–397, and 405–441) and L50 (residues 467–597 and 638–661) domains. (C) MDFF indicates the Milligan contact cation-π interaction between R561 in the MD loop 3 and Y91 in the adjacent actin is absent in ADP (left) but is established upon transition to the rigor state (right), with clear density for these sidechains in the rigor map. For both states, all six actomyosin interfaces in the corresponding HR MDFF model are displayed superimposed on one actin subunit as described in Figure 2A. Density maps are displayed in transparent grey in the upper panels. Orange dotted circle indicates absence of density for R561 in the ADP map, while density for Y91 is still present.

Figure 3—figure supplement 1. Comparison of ADP and rigor motor nucleotide binding cleft.

(A) View of the MD from the LPF ADP MDFF model (left, dark magenta) and LPF rigor MDFF model (right, light magenta), rigid-body fit into their corresponding segmented density maps (transparent grey) low-pass filtered to 7.5 Å. (B) View of the boxed region indicated in panel A, comparing the myosin VI nucleotide binding cleft between ADP (left) vs. rigor (right). Top panels show density maps only; bottom panels include the rigid body fit models as described in A. Density corresponding to ADP nucleotide is orange.

Figure 3—figure supplement 2. Validation of ADP MDFF model.

Per-residue Cα RMSD is displayed between superpositions of backbone-averaged HR MDFF models. The superposition was generated based on the Cα coordinates of the indicated full motor domains. The backbone of the first state indicated is displayed and colored. Differences of the lowest magnitude occur between ADP from 2BKI vs. ADP from 4PFO.

Figure 3—figure supplement 3. Interactions composing the actomyosin VI interface in the ADP state.

(A) All six actomyosin interfaces from the HR ADP MDFF model, superimposed based on the Cα coordinates of actin protomer 1 (dark-blue), as described in Figure 2A. MD, dark magenta; actin subunits, varying shades of blue. (B–F) Detailed views of interface contacts suggested by MDFF, colored as in A. EM density map is displayed on left side in transparent grey. (B) Hydrophobic interaction between MD HLH and actin SD1/SD3. (C) Milligan contact interactions between MD loop 3 and actin D-loop/SD1. (D) Electrostatic interaction between MD loop 4 and actin SD 3. (E) Interface of MD HCM loop and loop 2 with actin surface colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). (F) Salt bridge formation between the base of the MD HCM loop with actin SD1.

Figure 3—figure supplement 4. Comparison of interactions composing the actomyosin VI interface between ADP and rigor.

(A) All six actomyosin interfaces from the HR ADP MDFF model as described in Figure 3—figure supplement 3. MD, dark magenta; actin subunits, varying shades of blue. (B–F) Detailed views of interface contacts, colored as in A; HR rigor MDFF model, panels from Figure 2B–2F, displayed on left side for comparison. (B) Hydrophobic interaction between MD HLH and actin SD1/SD3. (C) Milligan contact interactions between MD loop 3 and actin D-loop/SD1. (D) Electrostatic interaction between MD loop 4 and actin SD 3. (E) Interface of MD HCM loop and loop two with actin surface colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). (F) Salt bridge formation between the base of the MD HCM loop with actin SD1.

Figure 3—figure supplement 5. Transducer rearrangement comparisons between myosins V and VI.

Transducer rearrangements between ADP and Rigor. Top panels: View of the transducer from the LPF ADP MDFF model (right, purple) and LPF rigor MDFF model (left, magenta), rigid body fit into their corresponding density maps. Bottom panels: Comparison of the myosin VI (left) and myosin V (right) transducer. View of the superposition of LPF ADP MDFF (purple) and LPF rigor MDFF (magenta) models, and Myosin V ADP (EMDB 31289, dark green) and rigor (EMDB 31288, green) structures. To generate these superpositions, the ADP and rigor density maps were aligned, then their corresponding atomistic models were rigid body fit into the aligned maps.

Figure 3—figure supplement 6. Milligan contact comparisons between myosins IIC, V, and VI.

(A) MDFF indicates the Milligan contact cation-π interaction between R561 in the MD loop three and Y91 in the adjacent actin is absent in ADP (left) and ADP from 2BKI (right) but is established upon transition to the rigor state (middle). For all three states, all six actomyosin interfaces in the corresponding HR MDFF model are displayed superimposed on one actin subunit as described in Figure 2A. (B) Myosin V R542, analogous to R561 in myosin VI, does not form a cation-π interaction with actin Y91 in ADP (left, dark green) or rigor (right, green). Actin is displayed in dark grey. (C) Myosin IIC (light pink, PDB 5JLH) Milligan contact does not form a cation-π interaction with (non-muscle) actin Y90 (grey).