Fig. 2. Co-translational folding of FLN5 monitored by ¹⁹F NMR spectroscopy.

a, Design of FLN5 RNCs in which FLN5 is tethered to the PTC via a linker sequence comprising a variable number of FLN6 residues and an arrest-enhanced SecM stalling motif. b, Anti-hexahistidine western blot of purified FLN5 RNCs, with and without ribonuclease A (RNase A) treatment. Representative data shown from two independent repeats. c, The ¹⁹F NMR spectra of FLN5 RNCs with increasing distance from the PTC. Observed spectra shown in grey were fitted and peaks assigned to U, I1, I2 or N states (coloured), with the sum of the fits shown in black. NMR data were multiplied with an exponential window function (10 Hz line broadening factor) before Fourier transformation. d, The ¹⁹F NMR spectrum of FLN5 + 34 RNC, processed with a line broadening factor of 5 Hz. Residual spectrum after fitting is shown below. e, Folding of FLN5 on the ribosome, measured using ¹⁹F NMR line-shape fits. f, Line-widths measured by line-shape fits of spectra as shown in c. All error bars indicate errors calculated by bootstrapping of residuals from NMR line-shape fittings.

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