Figure 5. SAXS-based shape reconstruction of GCaMP2 in the presence or absence of Ca²⁺.

(A) Size exclusion chromatographic (SEC) analysis of GCaMP2 in the presence and absence of Ca²⁺. GCaMP2ΔRSET was purified in its monomeric state (see Material and Methods for details). Proteins were analyzed on a S200 gel filtration column (10/300; GE Healthcare) in gel filtration buffer containing EGTA (10 mM; orange trace) or Ca²⁺ (1 mM; green trace). (B) Solution scattering data for GCaMP2ΔRSET in its Ca²⁺-bound and Ca²⁺-free state. Small-angle X-ray scattering curves of GCaMP2ΔRSET•Ca²⁺ (green) and its EGTA-treated form (orange) are shown after averaging and solvent-subtraction. Theoretical scattering profiles calculated from the ab initio models with the lowest χ values are shown (black line). The inset shows Guinier plots (including linear fits) at the low angle region (S_max*R_g<1.3). (C) Distance distribution [P(r)] functions for GCaMP2ΔRSET. P(r) curves of GCaMP2ΔRSET•Ca²⁺ (green) and its EGTA-treated form (orange) were calculated from SAXS data shown in (B) or from the crystal structure of GCaMP2ΔRSET•Ca²⁺ (dashed line). (D) SAXS-based shape reconstruction of GCaMP2ΔRSET•Ca²⁺. The overall volume from shape reconstructions after averaging (grey envelope) was calculated from 40 independent models. The crystal structure of monomeric GCaMP2ΔRSET•Ca²⁺ was docked into the envelope manually. Two orthogonal views are shown. (E) SAXS-based shape reconstruction of Ca²⁺-free GCaMP2ΔRSET. Details are as described in (C). The crystal structures of cpEGFP and Ca²⁺-free CaM (PDB code: 3CLN) (Babu et al., 1988) were docked into the envelope manually.