NMR measurements
Backbone amide group NH RDCs have been recorded and evaluated as described in (Lakomek et al. 2008a). For methyl group CH couplings, four constant-time 13C,1H-HSQC sub-spectra with varying 1JCH(+1DCH) evolution delays (Δ1 = 0, 1/6J, 1/3J, 1/2J) were recorded in an interleaved manner using the pulse sequence described by (Kontaxis and Bax 2001). Typically, each experiment included 89 complex increments in the indirect 13C dimension for the sampling of a 16-ppm sweep width, centered at 20 ppm. All experiments were recorded either on a 600 MHz DRX Bruker instrument equipped with a 5-mm H/C/N-probehead or on a 700 MHz Avance Bruker instrument equipped with a 5 mm H/C/N-probehead.
Sub-spectra were separated, processed and co-added to yield four spectra with single peak components corresponding to the ααα, ααβ/αβα/βαα, αββ/βαβ/ββα, or βββ coherence of each methyl group. Couplings (1JCH and 1JCH + 1DCH) were evaluated from the separation between the four components; for precise measurement, a dedicated FELIX script was designed to perform the spectral shifts necessary to simultaneously overlay the four components. Uncertainties on the couplings have been estimated as the range of shifts for which 4-peak overlays was acceptable. Assuming fast axial reorientation, each measured methyl CH RDC (1DCH3) was rescaled to the methyl CC RDC (1DCC), corresponding to the CC bond co-linear to the rotation axis, using a universal factor of −0.3155 according to (Ottiger and Bax 1999).