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. Author manuscript; available in PMC: 2020 Apr 18.
Published in final edited form as: Cell. 2019 Apr 4;177(3):751–765.e15. doi: 10.1016/j.cell.2019.03.012

Figure 2. Recombinant systems for PFD and XL/MS arrangement.

Figure 2.

(A) Purification strategies for human and yeast PFD. (B) SDS–PAGE of purified PFD complexes (Figure S2D). (C) Autoradiogram of NPAGE of yPFD and hPFD bound to denatured [35S]actin. (D) SDS–PAGE and Native-PAGE of hPFD −/+ DSS crosslinking. (E) Obtained XLs mapped onto homology models of hPFD and yPFD (solid black lines). (F) Histogram of number of XLs satisfied for every PFD subunit arrangement, the two arrangements satisfying the most constraints are mirror images in which each subunit has the same neighboring subunits. (G) Histograms of Cα-Cα distance of XLs mapped onto homology models of the two subunit arrangements that satisfied the most constraints. (H) Example immunoblot of serially diluted PFD (4–.06 μM) bound to TRiC (0.25 μM). (I) Quantification of immunoblots of TRiC-bound vs free PFD establish apparent dissociation constant, error bars represent SEM. (J) Role of ATP in TRiC–PFD interaction: Measured relative rotational diffusion (free Alexa647-PFD set to 1) of Alexa647-PFD mixed with TRiC ATP-AlF4 (closed), TRiC no nucleotide (open), and absence of TRiC.