Supplementary Figure 3: Ci-VSD WT structure details at 3.6-Å resolution.

(a) Ci-VSD WT+39D10_18 crystal belongs to space group P1 and contains 4 complexes (2 dimers) per asymmetric unit. B. Backbone of Ci-VSD WT. (b) Cα is shown in sphere for the S4 arginines (blue to cyan) hydrophobic gasket (yellow) and counter charges (red). (c) 2F_o-F_c electron density maps (σ=1.0) of one representative WT+39D10_18 complex. The S4 segment and S4-phosphatase linker were colored in red. The backbone of Ci-VSD was well resolved. (d) Superposition of the four copies of the Ci-VSDWT+39D10_18 complex inside the P1 unit cell. Backbones for the three individual domains were aligned separately and overlapped with each other within the four copies (left). Dramatic differences showed up among the four complexes when they were aligned at Ci-VSD as a whole unit (right). The variation in the four variable domains is only 1~2 Å, but extends to ~12 Å among constant domains. This apparent flexibility comes from the relative position between individual structural domains, particularly between the constant and variable domains of the Fab. The individual transmembrane regions of Ci-VSD WT are shown to be essentially identical when superimposed. There is clear heterogeneity of S4-phosphtase linker in Ci-VSD-WT since it is resolved in only three out of four copies in the asymmetric unit (e), even though the interactions involved in S4 are the same among four copies.