Extended Data Fig. 3: Nanobody Nb18 characterisation.
From: Snapshots of actin and tubulin folding inside the TRiC chaperonin
(a) Nb18 (red box) pulls down overexpressed CCT5 (left, n = 2) and endogenous TRiC (right, n = 1) from affinity chromatography. (b) Nb18 binds recombinant CCT5 in biolayer interferometry (Kd = 86 nM). Inset: Raw sensogram showing that recombinant CCT4 does not bind Nb18. (c) CCT5 and TRiC ATPase activity in the presence of Nb18. Nb18 was added at 5:8 molar ratio to recombinant CCT5, and at 1:1 to endogenous TRiC. Data are presented as mean values +/− SD (n = 6 for CCT5 only, n = 3 for all other samples). (d) Nb18 binds to a hydrophobic interface of CCT5 in proximity to ATP binding pocket. (e) Interacting residues between Nb18 and CCT5 in closed-TRiC (left) and open-TRiC (right). Green dashed lines indicate van der Waals contacts and blue dashed lines indicate hydrogen bonds. (f) Structural representation of CCT5-Nb18 crosslinks. Crosslinked residues are highlighted as spheres in gold. Dotted lines indicate distances between amines. Uncropped gels in a are available as source data.
