Figure 1
From: Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein
Scaffolding strategy and biophysical screening of MBP-GS fusion constructs.
(A) Scaffolding strategy. Top: The target monomer (magenta) is fused via a linker (green) to the scaffold protein (blue). Boxes represent the minimal regions required for proper subunit folding and oligomer assembly. Bottom: The junction length is optimized to bring the structured domain(s) of the target into contact with the scaffold and thereby yield a rigid, symmetric particle. (B) MBP-GS fusion constructs and their junction sequences. (C) Ribbon diagram illustrating the fusion of MBP to GS. The MBP N-terminal domain (NTD) is in magenta and the C-terminal domain (CTD) in pink. The 2 C-terminal MBP and 12 N-terminal GS residues dispensable for fold stability and oligomerization are in purple and navy blue, respectively. (D) Native PAGE analysis of MBP-GS fusion constructs. (E) Biophysical analysis of constructs A3 to Δ8. The left panels summarize results for all 12 constructs. The right panels show raw data for 3 representative constructs (circled in green, blue and magenta) that illustrate the spread of values observed in each assay. Top, Summary of hydrodynamic radii (Rh) determined by SEC, with representative chromatograms shown at right. Middle, Plot of polydispersity index determined by DLS, with profiles illustrated at right. Bottom. Summary of melting temperature (Tm) determined by DSF, with representative thermal denaturation curves shown at right. See also Supplementary Figs S1–S3.
