Extended Data Fig. 1: Small Angle X-Ray Scattering indicates that Sas6 remains mostly compact in solution with minor extension beyond that of the crystal structure.
a. Total subtracted scattering intensity (left y axis) and Rg (right y axis) as a function of time for the SEC-SAXS elution. The elution resolved several peaks, including a single strong monodisperse peak as indicated by the constant radius of gyration (Rg). b. Guinier fit analysis with normalized residual shown in the bottom panel. Rg and I(0) values of 29.44 ± 0.04Å and 0.04 ± 3.65 × 10−5 were obtained and the fit and normalized fit residuals confirmed this peak was monodisperse. The molecular weight of Sas6T from the SAXS data was calculated to be 61.0 kDa (theoretical 68.9 kDa) indicating it is primarily monomeric in solution. c. P(r) versus r normalized by I(0). The Dmax from the P(r) function for Sas6T is 90Å. The overall shape of the P(r) function for Sas6T, calculated by indirect Fourier transform (IFT) using GNOM, has a relatively Gaussian shape that is characteristic of a globular compact particle with the main peak at r = ∼30 Å. There is a small peak at r = 55Å which suggests there are two structurally separate motifs, possibly RbCBM26 and RbCBM74. d. Dimensionless Kratky plot; y = 3/e and x = \(\sqrt{3}\) as dashed gray lines to indicate where a globular protein would peak. The small plateau in the mid to high q region, around qRg = 5 in the dimensionless Kratky plot indicates some extension or disorder in the system. These results suggest the presence of two separate modules with flexibility between them, likely corresponding to the two CBMs. e. FoXS and f. MultiFoXS fits (black) to the Sas6T SAXS data (red) with normalized residual shown in the bottom panel. The FoXS fit had a χ2= 2.46 and showed systematic deviations in the normalized fit residual suggesting significant differences between the lowest energy conformation of Sas6T in the crystal structure and the structure of Sas6T in solution. For MultiFoXS we assigned the linkers between the domains (residues 130-137 and 572-583) as flexible. MultiFoXS gave a best fit with a 1-state solution with a χ2= 0.96 and calculated Rg of 29.2Å which corroborates the Guinier Rg calculation. g. Topology map of BIgA and BIgB domains illustrating the Greek key motif in BIgA and showing the loops that hydrogen bond with one another. h. A surface area analysis of the BIg domains using PISA in CCP4 gives a buried surface area of 353.9Å34. Residues providing hydrogen bonding are represented by stick side chains and the hydrogen bonds are shown by dashed yellow lines.
