Concerning the focus of this study - the relative frequency of chalcogen and hydrogen bonds – it is interesting to observe that hydrogen bonds are about 40-50 times more common than chalcogen bonds (Table 1 ). This suggests that they are stronger. Not 40-50 times stronger, obviously. This indicates that in most of the cases, the hydrogen bond that a thiol group may form is more stable that alternative chalcogen bond: even a small difference would produce the prevalence of hydrogen bond, from a thermodynamic perspective. Precise estimations of the energy of these interactions is unfortunately impossible, based on statistical observations, since the probability density functions of the energies are unknown. However, it is clear that hydrogen bonds are stronger, on average.

Table 1 also shows that trends and tendencies evaluated by using a non-redundant subset of the Protein Data Bank (Singledataset) or by following the RaSPDB method (raspdb_x datasets) are nearly equivalent. This reinforces the use of the RaSPDB method, which allows one to use a greater amount of information and to compute estimated errors.

The trends outlined above are independent on the secondary structure or on the degree of solvent accessibility of the cysteines.

If chalcogen and hydrogen bonds would have the same strength, one would expect two-to-three hydrogen bonds per chalcogen bond, since the number of hydrogen bonds that a cysteine thiol group can form is likely to be higher than the number of chalcogen bonds that it can form (Figure 1 ). However, the observed difference in the number of bonds is much higher.

The interaction energy of both chalcogen and hydrogen bonds can be quite variable. Both chalcogen and hydrogen bond have an electrostatic component, which strongly depend on the local environment – i.e.on the local dielectric constant. Consequently, chalcogen bonds may be stronger than hydrogen bond, in some cases.

It is interesting to observe that the presence of a hydrogen donor may hinder, because of steric reasons, the formation of a chalcogen bond along the extension of the C-S covalent bond. In other words, the hydrogen donor and the nucleophile roughly compete for the same position close to the sulfur atom (Figure 1 ).