Structural constraints in consistent nucleosomes reflected in SymCurv values. (A) Distributions of SymCurv values for 1,099 consistent nucleosome-free (NFR) sequences, 3,061 consistent nucleosomes and 3,288 weakly positioned nucleosomes in the form of box plots. One value was calculated for each sequence as the average SymCurv value over all nucleotides (see Methods). Solid boxes extend to cover the core 50% of the values around the median, whiskers extend to the further points, and notches extend to 1.58 of the interquartile range. Non-overlapping notches between two distributions suggest significant differences at 95% confidence interval. Values of consistent nucleosomes are significantly greater than both linker (t-test, P < 10-16,) and weakly positioned nucleosomal sequences (t-test P < 0.001). (B) Normalized SymCurv values for the central 447 nucleotides of consistent against weakly positioned nucleosomes. Both forward and reverse complements of each sequence were included in the analysis. Values were calculated here as the average SymCurv value over each position on a set of centrally aligned sequences (that is, the centre of the sequences was set to 0, and each nucleotide was assigned a coordinate relative to the centre), and were subsequently normalized against the overall average in the yeast genome (see Methods). Curves were smoothed with a running average on a window of 50 bp for better representation. Dotted vertical lines represent the boundaries of consistent nucleosomes. (C) Robustness of SymCurv values for consistent NFR, consistent nucleosomes and weakly positioned nucleosomes. Robustness was defined as the negative logarithm of average SymCurv variance in the set of one-nucleotide neighbors of each sequence. Low SymCurv robustness values for consistent nucleosomes versus weakly positioned nucleosomes (t-test P < 10-7) reflect increased constraints at the structural level. Similarly low values of consistent NFR sequences suggest that such constraints are also present in these sequences apart from those at the sequence level.