Proposed equilibrium link between dimer and tetramer of the hcSHMT/Δflap. The equilibrium between apo-dimer and holo-tetramer in hcSHMT/Δflap upon addition of PLP can be explained by the diagram. Path A, PLP can bind to the apo-dimer to form a holo-dimer, which is finally assembled to the holo-tetramer. Path B, PLP can be dissociated from the holo-tetramer to give the apo-tetramer with a KdPLP value of 0.228 ± 0.005 μm. High thermal motion (high B-factor) was found in the apo-tetramer, indicating high probability of dissociation of the apo-tetramer to a more stable apo-dimer (low B-factor). Upon increasing the PLP concentration, the binding of PLP promotes a shift toward holo-tetramer stabilization, possibly by more tightly packing the domains (indicated by the relative B-factors). The relative B-factors were calculated from MD simulations as described under “Experimental procedures.”