Fig. 5: Characterization of RTR homotetramers and hetero-oligomers.

a Cartoon representation of three representative successful redesigns of RTR0. Blue and purple colored backbones represent N- and C-terminal interfaces, respectively. HBNets and disulfide bonds are shown in sticks with hydrogen bonds in dashed lines. Design names (bottom left corner of each box) and box outline and trace colors are used consistently throughout to represent the same interfaces. Characterization of the three examples from (a) by (b) SEC, c nMS, d SAXS, and (e) nsEM. b Overlay of A280 normalized SEC traces. c Cropped view from one of 173 micrographs of RTR05 collected from a single sample. d Individual 2D class averages of nsEM data collected on each sample. The outline color corresponds to the sample ID. e SEC traces (normalized absorbance at 230 nm) of four hetRTRs: (left to right) A2B2-hetRTR05-11_noCys, A₅2B₇2-hetRTR05-11_noCys, A3B3-hetRTR05-11_noCys, and A2B2C2-hetRTR11_noCys-05-16. Inset with cartoons representing the expected oligomer shapes. Shading indicates the region used for nsEM analysis. f Pairs of 2D class averages matching the SEC traces above, except A₅2B₇2-hetRTR05-11_noCys which shows one 2D class average and an overlay of the 2D class with a 2D class of RTR11 to show the angular shift.