Fig. 1: Design and characterization of Rigid Fabs. | Nature Communications

Fig. 1: Design and characterization of Rigid Fabs.

From: Disulfide-constrained Fabs overcome target size limitation for high-resolution single particle cryoEM

Fig. 1

A Design of Rigid Fabs. To engineer Fabs that are conformationally rigid, two (2DS) or four (4DS) intrachain disulfides were introduced in the elbow region of both the light (gray) and heavy (yellow) chains to restrict the elbow angle between the variable and constant domains. Two additional interchain disulfides were introduced in the constant domain to further reduce the flexibility of this domain, leading to a Rigid Fab design containing six engineered disulfides (6DS). B Crystal structure of E104.v1.6DS Fab. Center, cartoon representation of E104.v1.6DS (HC, yellow; LC, gray) with disulfides shown in spheres (2DS disulfides, cyan; 4DS disulfides, blue; 6DS disulfides, pink). Insets show electron density contoured at 1σ for each of the engineered disulfides. Maps of tryptase complexes with C WT, D 2DS, E 4DS, and F 6DS variants of the E104.v1 Fab colored by local resolution. EM density for selected map regions illustrating improvement in resolution with increasingly rigid constructs of E104.v1: G–J tryptase P84-S86 (green sticks) and K–N E104.v1 HC A31-A34 (yellow sticks) from the indicated tryptase-Fab structures.

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