Figure 3
From: Structural analysis of biological targets by host:guest crystal lattice engineering
Design of constructs L1 and L2. (A) Overlay of three DARPin-domain orientations viewed along the shared helix. The EngBF auxiliary domain is shown as a grey surface and DARPin rotations 4, 6 and 9 are highlighted as cartoons in pink, blue, and orange, respectively. Rotations were generated by stepwise extension of the helix linker between the EngBF and the DARPin-domain. (B) Alignment of linker helices that are shared between EngBF and target-binding domain. (C) Experimental structure of EngBF_DARPin_rot4 (light blue and orange cartoon) superimposed on the design model (dark green cartoon, the EngBF domain has been omitted for clarity). The 2mFo − DFc map was contoured at 1 σ showing partial density for the C-terminal DARPin. In the experimental crystal structure, the three-helix bundle was shifted, causing a partial unwinding of the connecting helix and a 120° rotation of the DARPin domain. (D) Overview of inserted disulfide bridges. The EngBF-DARPin fusion is shown as a Cα-trace with the EngBF domain in grey and the DARPin domain in light blue (construct L1) and green (construct L2). Symmetry-related molecules are shown as molecular surfaces and disulfide bridges as spheres. Termini and disulfide bridges are labelled.
