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Table 1. Root mean square (RMS) deviation values, characterizing conformationalclusters of 1 and 2 and the differences between both fitting approachesLES-MD/AMBERconfidence level 0.8EDMC/ECEPP [1]confidence level 0.94AMBER vsECEPP1No of structures: 154/23(α=0.001/α=0) 7(α=0) 154/7aσ J 0.011/0.003 0.88σ b NOE 0.13/0.13 0.32RMS c : All 2.33/2.27 1.22 2.52Macrocycle 1.02/1.01 0.66 1.312No of structures: 26/3(α=0.001/α=0) 11(α=0) 26/11σ a J 0.007/0.003 0.78σ b NOE 0.05/0.05 0.18RMS c : All 2.40/2.01 1.79 2.46Macrocycle 0.96/0.91 0.77 1.49ain Hz; b unitless; c ”heavy”C, N atoms, in Åfor 1 and 26 for 2, restoring the NMR parameters to about 80%. The value of α=0.001certainly have not eliminated overfit (e.g. 3 J NCα -s were restored to an astonishing apparentaccuracy of ~0.01 Hz, see Table 1), yet, mere introduction of α has dispersedconformational variability from 23 to 154 for 1, and from 3 to 26 for 2, as referred to α=0(Table 1). The representative structures of 1 and 2 are shown in Figure 1. The RMSdeviation values calculated using all the heavy atoms and those of the restraining rings (seeabove) are shown in Table 1.In agreement with the work of Groth, at al. [2], the overfit falls down rapidly with theintroduction of even the slightest share of entropy to the objective function (comp. higherRMS for α=0.001 than for α=0, Table 1). Intriguing is startlingly small σ J ≈0.01 Hz atα=0.001 for LES-MD/AMBER methodology, as compared to σ J =0.8-0.9 Hz forEDMC/ECEPP at α=0. A similar feature is not the case when comparing σ NOE in bothmethods. Hence, at minute entropy contributions, it follows that J does or does nottranspose “overfitted” into the final SD in the AMBER or ECEPP ensemble, respectively.This is apparently an effect of softness of the AMBER vs ECEPP force field and its effecton ensemble sampling. A further corollary is that using AMBER ensemble for fittingflexible peptides, J restraints may be omitted with a minute loss in quality of fit. As NOEcontributes to structure determinations from NMR in a major way while 3 J NCα is lesssignificant [4] this is not a concern and comparisons between equilibria resultant fromECEPP [1] and AMBER are justified. Thus, both methodologies give similarconformational equilibria, in which the restraining rings fit well within 1,31Å for 1 and1,49Å for 2 (Table 1). Apparent differences seen in Tyr and Phe (Figure 1) arise fromconformational freedom of their side chains.AcknowledgmentsThis work was supported by Polish Ministry of Science grant DS 8360-4-01330-0. The calculationswere carried out in the Academic Computer Centre (TASK) in Gdańsk, Poland.References1. Filip, K., et al. J. Peptide Sci. 11, 347-352 (2005).2. Groth, M., et al. J. Biomolec. NMR 15, 315-330 (1999).3. Case, D.A., Darden, T.A., Kollman, P.A. AMBER8 Manual, University of California, San Francisco;and references therein (2004).4. Rule, G.S., Hitchens, T.K. Fundamentals of Protein NMR Spectroscopy, Springer 2006, Chapter 27.599