Extended Data Fig. 3. Biochemical and NMR analysis of aromatic RNC variants.
(a) Anti-His tag western blot of FLN5+31 wt, A3Y3, F3A3 and A3A3 RNCs (10 pmol each) purified for NMR studies, in their tRNA-bound and released forms. The migration patterns on SDS page reflect those of the isolated mutant proteins (cf. Extended Data Fig. 2). (b–e) 1H,15N SOFAST-HMQC spectra (283 K, 950 MHz) of (b) FLN5+31, (c) FLN5+31 A3Y3, (d) FLN5+31 F3A3 and (e) FLN5+31 A3A3 RNCs against the corresponding isolated, unfolded FLN5 variants, with assignments. (f–i) Assessment of the integrity of 15N-labelled RNCs25: Anti-His western blots and translational diffusion coefficients measured using 15N SORDID experiments are shown. The timeframes during which nascent chains have been assessed as being intact and used for analysis are indicated (dashed boxes). Vertical error bars represent standard errors derived from the spectral noise; horizontal bars (where applicable) indicate the acquisition periods of diffusion measurements.