Figure 1.

Principle of LEGO-NMR spectroscopy: A) shown is method A1 (Table S2, Figure S1). In stage 1 the E. coli cells are grown in NMR active medium and protein synthesis is induced from plasmid 1, resulting in the production of NMR active LSm5, LSm6, and LSm7. In stage 2, the cells are transferred to an NMR inactive medium, where protein synthesis from plasmid 2 is induced, resulting in the production of NMR invisible LSm2, LSm3, LSm4, and LSm8. By addition of a purification tag to a single subunit of the complex, the intact complex can be straightforwardly isolated and purified to homogeneity. NMR active subunits are colored, whereas NMR invisible subunits are in gray. B) ¹H-¹⁵N TROSY NMR spectrum (gray) of the uniformly NMR active LSm2–8. Especially the central region suffers from severe resonance overlap, complicating spectral analysis significantly. C) Top left: LEGO ¹H-¹⁵N NMR spectrum (black) of the LSm2–8 complex, in which LSm5, LSm6, and LSm7 are NMR active and LSm2, LSm3, LSm4, and LSm8 are NMR invisible. Other panels: LEGO ¹H-¹⁵N NMR spectra of the LSm2–8 complex in which individual LSm subunits are NMR active in an otherwise NMR inactive background. The resonance signals in the spectra display a subset of the resonance signals observed in the fully labeled complex (see enlargements).