MF-derived side chain dynamics The order parameters derived from the average spherical harmonics obtained from methyl group RDCs measured in 13 different alignment conditions according to the MFA are plotted in Fig. 2. Of the 50 methyl groups in ubiquitin, 37 gave rise to order parameters and MF parameters. Residues for which RDC data was too sparse, as indicated by a small 5th eigenvalue (<0.35) of the F-matrix, were rejected. This affected the following residues: Thr7(Cγ1), L15(Cδ1), V17(Cγ1), I23(Cδ1), V26(Cγ2), I30(Cδ1), L43(Cδ1 + Cδ2), Thr55(Cγ1), L56(Cδ1), Thr66(Cγ1), L69(Cδ1), and L71(Cδ1). Dynamic Q-values have been calculated (see Supplementary Information, Table S1). The average dynamic Q-value is  = 0.13. Fig. 2 Methyl group a order parameters, and b amplitude of anisotropy, ηrdc measured from RDCs plotted against residue number in human ubiquitin. Also encoded in the graph are residue type (letter), their solvent exposure (grey core exposed; white solvent exposed) and the distance of the methyl group to the backbone (blueCβ; greenCγ; orangeCδ; redCε). RDC-based order parameters have a mean value of 0.43 with a standard deviation of 0.25 The range spanned by the methyl axial order parameters shows striking heterogeneity in methyl group dynamics. They vary from very rigid (e.g.,  ~ 1 for A28 (Cβ), I44(Cγ2) and A46(Cβ) to very mobile [e.g.,  ~ 0.05 and 0.1 for L69(Cδ1) and L8(Cδ1)]. The mean order parameter over all measured methyl groups in ubiquitin is with a standard deviation of σ = 0.25. This value is much lower than the average order parameter measured on the more rigid backbone NH groups on the same protein and under the same conditions (<(NH)> = 0.72 ± 0.02; Lakomek et al. 2008a). The magnitude of errors is larger for methyl groups than for NH backbone amide groups (errors for DNH were estimated to be 0.3 Hz or less). The first reason is a limited resolution of the RDC measurements using a constant-time HSQC (Kontaxis and Bax 2001) with the limited maximum evolution time corresponding to approximately 15 Hz resolution compared to better than 2 Hz resolution for NH groups measured on a normal HSQC (without zero filling). Secondly, the coverage of the five-dimensional RDC space is less complete than for the NH measurements, as indicated by a lower 5th eigenvalue of the F-matrix. Although the combined effect of both errors propagates, a quantitative analysis of the results was still possible. Three notable order parameters have values larger than 1, based on the MF theory. Those correspond to methyl groups of A28 (Cβ), I44(Cγ2) and A46(Cβ). Whereas such values are impossible in reality, they are still within experimental error in the allowed range for order parameters (0–1). For statistical purposes, these three order parameters were corrected to the maximum physical value of 1. In Fig. 2b, the MF parameter ηrdc is shown for each methyl group in ubiquitin. This parameter represents the anisotropy of motion, assuming values from 0 (perfectly axially symmetric motion) to √¾ (fully anisotropic). This parameter, in combination with the order parameter, is useful for differentiating complex types of motions; for example, deciphering those dominated by two or three rotameric jumps or by libration motion. Also for the amplitude of anisotropy ηrdc, a large heterogeneity is observed, with variations seen from 0.04 ± 0.05 (V17(Cγ2)) to 0.83 ± 0.15 (L71(Cδ1)).