Backbone conformational preferences are a common method of validation for force field development because they play a key role in determining the secondary structure preferences of simulated proteins. Here we examined the backbone conformational preferences of all nine dipeptides, using the same definitions of secondary structure (i.e., "helical", "PPII", and "extended") as Vymětal et al. to facilitate direct comparison with that work and additionally validate our own simulation results. We classify φ/ψ angles not corresponding to one of these three regions as "other"; such conformers would be considered as either αL or αr in Vymětal et al. As in that work, we find that phosphorylation and then deprotonation of the phosphate group successively increase the helical nature of the serine dipeptide when simulated using ff99SB (Figure ). Likewise, phosphorylation causes a modest shift from PPII to extended conformations in threonine, whereas the opposite trend appears for tyrosine (Figure ). With FB18, however, the shifts in conformational preferences are generally much larger, especially for the deprotonated phosphorylated residues. In particular, we see that deprotonated phosphorylated serine (SEP) has more helical content and greatly reduced extended and PPII content compared to neutral serine. (The missing ∼50% of conformers are in the lower left of the α-helical (αr) region of the Ramachandran diagram and are therefore classified as "other" rather than "helical" according to the definitions of these regions. ) The same is largely true for threonine under FB18, although more of the conformers remain in the "helical" region. Finally, for tyrosine we see that the deprotonated phosphorylated residue (PTR) has a much larger proportion of "extended" conformers than its neutral or protonated phosphorylated counterparts (Figure ). Indeed, it appears that the predicted impacts of phosphorylation on backbone conformational preferences are quite different between ff99SB and FB18. One way to begin to address which force field is more accurate is to examine experimental data that reports on these preferences; in this case, J HN,Hα scalar couplings report directly on the ensemble average of the φ angle distribution. Experimental 3 J HN,Hα scalar coupling data are available for both unmodified and phosphorylated (both protonation states) serine and threonine, but only unmodified tyrosine. As shown in Table and Figure , ff99SB generates scalar couplings that are close to experiment for unmodified residues and protonated phosphorylated serine (S1P) and threonine (T1P). But the predicted scalar couplings for deprotonated phosphorylated serine (SEP) and threonine (TPO) are far from the experimental values (∼1.5 Hz and ∼2.5 Hz, respectively). Interestingly, all of predicted scalar couplings generated by ff99SB reside between approximately 6.9 Hz and 7.6 Hz, regardless of residue identity, phosphorylation, or protonation state. This can be rationalized by noting that the backbone conformational preferences, specifically the proportions of extended conformers (φ centered on -165 • ) vs. proportions of helical + PPII conformers (φ centered on -60 • ), are quite similar for all of these dipetides under ff99SB. As with the backbone conformational preferences, FB18 generates predictions that are qualitatively different. In particular, FB18's predictions agree with experiment to a level of ≤ 0.5 Hz for all of the simulated dipeptides. To quantify the agreement between the scalar couplings predicted from our simulations and the experimentally measured couplings, we computed both the χ 2 score, as in Best et al., and the (Pearson) correlation coefficient r for these data. Across all seven residues for which there are experimental data, we found that the χ 2 score for ff99SB was 9.78, while the χ 2 score for FB18 was 0.79. If we restrict the comparison to only the phosphorylated residues with experimental data (i.e., S1P, SEP, T1P, and TPO), then the χ 2 scores for ff99SB and FB18 are 16.78 and 1.14, respectively. We note that a χ 2 score of 1 or below suggests that the predicted couplings are comparable to the experimentally measured couplings, given all of the uncertainties present. Calculating the correlation coefficient r and its 95% confidence interval (CI) for the scalar coupling data of all residues under ff99SB yields -0.293 (95% CI: