Extended Data Fig. 1: Labeling strategy for single molecule imaging of TRiC, PFD and ribosome.
From: Single-molecule dynamics of the TRiC chaperonin system in vivo
a, CRISPR-Cas9 mediated knock-in of a Halo-tag at a loop region between S202 and V203 of CCT4. Structures of CCT4 in cartoon model and of TRiC in surface map (PDB:7X6Q) are shown with the site of insertion indicated. b, CRISPR-Cas9 mediated knock-in of Halo-tag at C-terminus of PFD4. The structure of PFD (PDB:1FXK) is shown in cartoon model with PFD4 highlighted. c, Immunoblot analysis of CCT4-Halo expressed in U2OS cells using anti-Halo antibody (left) and anti-CCT4 antibody (right). GAPDH served as loading control. d, Volcano plot representation of label-free proteome analysis of TRiC-Halo-tag pulldown fractions from cell lysate as in (c) (also see Supplementary Table 1a). e, Immunoblot analysis of PFD4-Halo using anti-Halo antibody (top) and anti-PFD4 antibody (middle). GAPDH served as loading control (bottom). f, Volcano plot representation of label-free proteome analysis of PFD-Halo-tag pulldown fractions from cell lysate as in (e) (also see Supplementary Table 1b). g, Construct of the large ribosomal protein L10A labeled with SNAP-tag (SNAP-tag-L10A). Immunoblot analysis of SNAP-L10A using anti-SNAP antibody (left) and anti-L10A antibody (right). h, Immunoblot analysis of soluble and pellet fractions from stable cell line expressing SNAP-L10A. i, Lysate from cells stably expressing SNAP-L10A was analyzed by sucrose gradient fractionation. UV absorbance at 260 nm was used to assess 40S, 60S, 80S and polysome traces (top). Immunoblot analysis of sucrose gradient fractions using anti-L10A and anti-SNAP-tag antibody (bottom) showing SNAP-L10A incorporation into polysomes similar to endogenous L10A. j, Normalized fluorescence intensity of cells endogenously expressing TRiC-Halo (left) or PFD-Halo (right) when incubated with fluorescent ligand JF646. “no significance (ns)” indicates the dye concentration at which the fluorescence signal reaches a plateau (n = 8 cells for each condition). k, In-gel fluorescence assay used to quantify the fraction of labeled TRiC-Halo (left) and PFD-Halo (right). To avoid saturation, 5-fold less protein was loaded for samples at plateau dye concentration than for those at the imaging concentration. Fluorescence from the gel (top) and total protein staining with Coomassie (bottom). l, Numbers of TRiC-Halo and PFD-Halo single molecules detected in the first frame of each video. Cell numbers [n]: TRiC-Halo, n = 10, PFD-Halo, n = 10. m,n, Co-movement trajectory of TRiC-ribosome (m) and PFD-ribosome (n) as shown in Fig. 1b and c, respectively. o,p, 1-cumulative distribution function (1-CDF) of interaction lifetimes of ribosome with TRiC (o) and PFD (p) were fitted with one-component decay models.
