Figure 3. Excited P-loop states are less compatible with nucleotide than the states preferred in crystal structures.

(A) The β-cardiac whole-motor MSM-derived P-loop conformational space projected onto PCs 1 and 3 reveals two distinct free energy basins (green level sets). Yellow and purple points represent crystal structures with and without ligand, respectively. Structures farther than 0.6 Å from the β-cardiac myosin structure (red empty circle) are labeled with their PDB ID. (B) Proximity to the β-cardiac myosin reference conformation is associated with the presence of a nucleotide in crystal structures (p<1.3×10⁻⁵ by Fisher’s exact test), suggesting that the ligand stabilizes the A state. Error bars represent the 95% confidence interval of 1000 bootstrap realizations. (C) The re-orientation of the S180 backbone carbonyl accounts for the split between upper and lower basins. Points represent P-loop conformations from each state in the β-cardiac whole-motor MSM projected onto the same PCs as in panel A. Points are sized by their probability from the MSM and colored by the angle between the backbone carbonyl bond vectors of S180 and K184. (D) Center, each of the five states of the P-loop MSM are indicated as nodes in a network, sized by their equilibrium probability and connected by arrows with line width proportional to the transition probabilities between them. Surrounding the model, insets show example configurations of the P-loop in sticks colored to match the state they represent. State A is associated with a conformation of the S180 (pink sticks) carbonyl bond vector (white arrow) directed away from the nucleotide binding pocket, whereas states B-D are associated with the opposite orientation of the S180 backbone carbonyl bond vector. The A state conformation is the conformation found in most crystal structures. For reference, PDB 1MMA is shown in grey sticks and the crystallographic position of ATP is shown in semi-opaque grey sticks. For all states, important interactions with the Switch-I loop are shown as two-dimensional sketches for visual clarity. An interaction between R237 and E179 is specific to state A, whereas various interactions with S242 are indicative of other states (Figure 3—figure supplement 2).

Figure 3—figure supplement 1. Variance explained by each component of the PCA of the of the P-loop on MYH7.

Each Principal Component (PC) in the PCA captures a certain fraction of the variance of the input data. Each PC is ranked by the amount of variance it explains. The above figure plots the PC number against the fraction of the overall variance it explains. Because 36 distances were used in total, 36 principal components are able to capture 100% of the variance. The top four PCs were used in clustering and for k-nearest neighbor state assignment.

Figure 3—figure supplement 2. Joint and marginal distributions for all pairs of PCs.

Each conformation in the whole-motor MSM has a value for each PC. Using these values and the probability for each conformation, a probability distribution for every individual PC’s value, and for every pair of PCs, were constructed. For MYH7, we visualized every marginal distribution (a single PC’s probability distribution) and every pair of PCs’ joint distribution. Diagonal plots, marginal distributions for PCs 1 (top left) through 4 (bottom right). Off-diagonal plots, joint distributions for all pairs of PCs; each column i and row j has PC i on the x-axis and PC j on the y-axis, respectively.

Figure 3—figure supplement 3. Weights of the first four principal components of the P-loop on MYH7.

The value of each PC for a particular conformation is a linear combination of each of the 36 input distances. In other words, it is the dot product of the vector of distances and the vector representing the PC (which weights each distance differently). This figure shows the relative contribution of each distance (x-axis) to each PC (y-axis). Percentages following principal component (PC) index indicate the fraction of the variance explained by that PC. Labels on the x-axis indicate a distance from [residue number][atom name] to [residue number][atom name], using the residue numbering found in the MYH7 crystal model 4PA0 (Winkelmann et al., 2015).

Figure 3—figure supplement 4. States of the whole-motor MSM of β-lactamase projected into PC1/PC2 and PC1/PC3 planes.

Each point, representing a single whole-motor MSM state, is colored by its P-loop state and sized according to its equilibrium probability. Note that, along the PC2 direction, the primary differences are between states C and E.

Figure 3—figure supplement 5. Specific interactions with Switch-I residues are statistical hallmarks of P-loop states.

Top left, the PDF over the distance between E179 sidechain carbonyl oxygens to the sidechain of R237. An interaction is a hallmark of the A state. Top right, the PDF over the S180O to S242H backbone-backbone distance. Close values are a hallmark of states B and E. Bottom left, the PDF over the E179 sidechain carbonyl oxygen to S242 amide proton distance. Low values are a hallmark of state C. Bottom left, the PDF over the distance between the G181 backbone carbonyl oxygen and the T185 backbone amide proton. Low values are a hallmark of state E. This is the i → i + 4 interaction typical of an alpha-helix.