Fig. 5. Visualizing the minor closed-state ensembles of CaM-4Ca²⁺ and apo CaM.

(A) CaM-4Ca²⁺-MLCK complex⁴ (with CaM in cyan and MLCK peptide in blue) overlaid on the CaM-4Ca²⁺ dumbbell structure³ (green), best-fitted to either the N-terminal (left panel) or C-terminal (right panel) domains. Twenty-six additional peptide-bound structures were overlaid in the same manner, and these are represented by the atomic probability density maps³⁵ shown in grey for the C-terminal domain (left) and N-terminal domain (right). These structures (PDB codes 1CDL, 1CDM, 1MXE, 1NIW, 1QS7, 1QTX, 1VRK, 1YR5, 1ZUZ, 2BE6, 2F3Y, 2F3Z, 2FOT, 2HQW, 2O5G, 2O60, 2VAY, 3BXL, 3BYA, 3DVE, 3DVJ, 3DVK, 3DVM, 3EWT, 3EWV, 3GP2) represent a wide array of different 1:1 canonical CaM-peptide complexes, but it can be seen that the conformational space occupied by the two domains is very similar to that in the CaM-4Ca²⁺-MLCK complex. (B–D) Atomic probability density maps showing the conformational space sampled by the minor species ensemble for (B) CaM-4Ca²⁺, (C) apo CaM, and (D) the apo CaM-MLCK complex. The minor state atomic probability maps are derived from 100 independent PRE-driven simulated annealing calculations using an 8-member ensemble (i.e. 800 total structures) at a population of 10%, and plotted at multiple contour levels ranging from 0.1 (transparent blue) to 0.5 (opaque red). The gray probability density maps, plotted at a single contour level of 0.1 of maximum, show the conformational space consistent with interdomain PRE values ≤ 2 s⁻¹ and represent the major state ensemble characterized by no interdomain contacts and an occupancy of ~90% (see also Fig. S9A). In the left-hand panels, all ensemble members are best-fitted to the N-terminal domain (dark green) and the probability density maps are shown for the C-terminal domain. In the right-hand panels, all ensembles are best-fitted to the C-terminal domain (dark green) and the probability density maps are shown for the N-terminal domain. All panels are displayed in the same orientation, and the extended dumbbell structure³ is displayed as a ribbon diagram for reference (with the structures of the individual domains in panels C and D replaced by those for apo CaM).