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\begin{document}

\title{Interplay between hydrogen and chalcogen bond in cysteine}



\author[1]{Oliviero Carugo}%
\affil[1]{Universita degli Studi di Pavia Dipartimento di Chimica}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Protein structures are stabilized by several types of chemical
interactions between amino acids, which can compete with each other.
This is the case of chalcogen and hydrogen bonds formed by the thiol
group of cysteine, which can form three hydrogen bonds with one hydrogen
acceptor and two hydrogen donors and a chalcogen bond with a nucleophile
along the extension of the C-S bond. A survey of the Protein Data Bank
shows that hydrogen bonds are about 40-50 more common than chalcogen
bonds, suggesting that they are stronger and, consequently, prevail,
though not always. It is also observed that frequently a thiol group
that forms a chalcogen bond is also involved, as a hydrogen donor, in a
hydrogen bond.%
\end{abstract}%



\sloppy


\section*{Introduction}

{\label{introduction}}

It was realized, long ago, that a folded protein ``consists of one
polypeptide chain which continues without interruptions throughout the
molecule (or, in certain cases, of two or more such chains)'' and that
``this chain is folded into a uniquely defined configuration, in which
it is held by hydrogen bonds'' {[}1{]}. Later on, a series of other
non-covalent interactions have been discovered to be responsible for
protein folding, stability, plasticity, and function, like van der
Waals, hydrophobic and electrostatic interactions {[}2{]}. One of these
non-covalent interactions has received little attention insofar: it is
the chalcogen bond.

It is an attractive interaction between chalcogen atoms (sulfur,
selenium or tellurium) and nucleophiles. In molecular moieties like
R-X-R (X = S, Se or Te), the nucleophile tends to occupy a position
along the extension of one of the R-X covalent bonds (\textbf{Figure 1}
){[}3{]}. Although recent publications reviewed extensively both
theoretical and experimental studies of chalcogen bonds {[}4{]}{[}5{]},
minor attention has been paid to this non-covalent interaction in
biological systems.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 1}. Scheme of the chalcogen and hydrogen bonds that may
involve the cysteine side-chain. \emph{Nu} indicates a nucleophile,
\emph{AA-A} a hydrogen acceptor, and \emph{D-H} a hydrogen
donor.\tabularnewline
\midrule
\endhead
\tabularnewline
\bottomrule
\end{longtable}

Despite a pioneering work of Thornton, reporting the interaction between
sulfur atoms of cysteine and methionine with the aromatic rings of
tryptophan, tyrosine and phenylalanine, which are nucleophiles {[}6{]},
little was published in the early days of structural bioinformatics. No
trace of chalcogen bonds involving methionine sulfur atoms emerged in a
1999 analysis of room-temperature protein crystal structures {[}7{]}. In
a subsequent study of a larger data set, some evidence of chalcogen
bonds between the methionine sulfur atom and backbone or side-chain
carboxylate oxygen atoms was observed {[}8{]}. Further statistical
analyses, coupled with molecular orbital \emph{ab initio} calculations,
confirmed that the sulfur atoms of cysteine and methionine can form
chalcogen bonds with protein polar atoms {[}9{]}{[}10{]}. More recently,
a 2021 study showed numerous chalcogen bonds between the selenium atom
of selenomethionine in low temperature protein crystal structures
{[}11{]}.

Ligand binding to proteins can be influenced by chalcogen bonds, too
{[}12{]}{[}13{]}. For example, the activity of ebselen, a glutathione
peroxidase mimic, is enhanced by its ability of forming chalcogen bonds
with selenium {[}14{]}. This non-bonding interaction is important also
in the mechanism of inhibition of maltase glucoamylase by salacinol and
katalanol {[}15{]}.

In this manuscript, the chalcogen bonds that involve cysteine
side-chains in proteins are identified and compared to the hydrogen
bonds that involve the same side-chains, which can behave either as a
hydrogen donors or as a hydrogen acceptors (\textbf{Figure 1} ). This is
an attempt to determine the relative energies of the two types of
interactions by comparison of their frequency of occurrence.

\section*{Results and Discussion}

{\label{results-and-discussion}}

All chalcogen and hydrogen bonds were identified according to the
procedures described in the Methods (see below). Interatomic contacts
that might be considered, according to the stereochemical criteria used
in this study, both hydrogen and chalcogen bonds were discarded (about
20\% of the chalcogen bonds may be confused with hydrogen bonds and
about 1\% of the hydrogen bonds may be confused with chalcogen bonds).

Details about hydrogen and chalcogen bonds are given in Supplementary
Material (\textbf{Tables S1-S4} ) and are only briefly presented here.

As it can be expected, given the high frequency of occurrence -- one per
residue -- the most common nucleophile that forms chalcogen bonds is the
main-chain oxygen atom. Surprisingly, sulfur atoms act rather frequently
as nucleophiles, too, despite their low frequency of occurrence in
proteins {[}16{]}. As it was already observed, cysteines tend to be more
frequently hydrogen donors that hydrogen acceptors in hydrogen bonds,
roughly with a ratio 2:1 {[}4{]}. When the cysteine thiol group acts as
a hydrogen donor, the hydrogen acceptor is often a main-chain oxygen
atom and when it acts as a hydrogen acceptor, the hydrogen donor is
frequently a main-chain or a water oxygen.

\textbf{Table 1}. Frequencies of chalcogen bonds (Cb) and Hydrogen bonds
(Hb) in the Single data set and in the nine subsets of the Protein Data
Bank assembled with the RaSPDB procedure (see Materials and Methods for
details). The average values (estimated errors in parentheses) are
computed on these nine subsets.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Dataset & Number Cb & Number Hb & \% Cb & \% Hb\tabularnewline
Single & 833 & 31972 & 2.54 & 97.46\tabularnewline
raspb\_1 & 321 & 16848 & 1.87 & 98.13\tabularnewline
raspb\_2 & 347 & 17141 & 1.98 & 98.02\tabularnewline
raspb\_3 & 359 & 17026 & 2.06 & 97.94\tabularnewline
raspb\_4 & 363 & 17255 & 2.06 & 97.94\tabularnewline
raspb\_5 & 346 & 16878 & 2.01 & 97.99\tabularnewline
raspb\_6 & 340 & 17288 & 1.93 & 98.07\tabularnewline
raspb\_7 & 359 & 16381 & 2.14 & 97.86\tabularnewline
raspb\_8 & 348 & 17171 & 1.99 & 98.01\tabularnewline
raspb\_9 & 347 & 16936 & 2.01 & 97.99\tabularnewline
Average-1-9 & & & 2.01(0.03) & 97.99(0.03)\tabularnewline
\bottomrule
\end{longtable}

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 (\textbf{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.

\textbf{Table 1} also shows that trends and tendencies evaluated by
using a non-redundant subset of the Protein Data Bank
(\emph{Single}dataset) or by following the RaSPDB method
(\emph{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
(\textbf{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 --
\emph{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 (\textbf{Figure 1} ).

\textbf{Table 2}. Fraction of chalcogen bonds associated with a hydrogen
bond involving the SG cysteine atom, in the Single data set and in the
nine subsets of the Protein Data Bank assembled with the RaSPDB
procedure (see Materials and Methods for details).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Dataset & Fraction (\%)\tabularnewline
Single & 66.3\tabularnewline
raspdb\_1 & 67.0\tabularnewline
raspdb\_2 & 64.6\tabularnewline
raspdb\_3 & 63.5\tabularnewline
raspdb\_4 & 66.7\tabularnewline
raspdb\_5 & 61.0\tabularnewline
raspdb\_6 & 65.0\tabularnewline
raspdb\_7 & 69.4\tabularnewline
raspdb\_8 & 69.3\tabularnewline
raspdb\_9 & 64.8\tabularnewline
Average-1-9 & 65.8(0.9)\tabularnewline
\bottomrule
\end{longtable}

Interestingly, the same thiol group of the cysteine side-chain can be
involved in both a chalcogen and a hydrogen bond (\textbf{Table 2} ).
About 65\% of the chalcogen bonds are associated with hydrogen bonds. An
example is shown in \textbf{Figure 2} : cysteine 31 (chain A) of human
thymidylate kinase forms a chalcogen bond with the main-chain oxygen
atom of alanine 37 and forms a hydrogen bond with the main-chain oxygen
atom of valine 27 (PDB file 1e9a, {[}17{]}).\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 2}. Example of chalcogen and hydrogen bonds formed by the
same cysteine thiol group (data from the file 1e9a of the Protein Data
Bank).\tabularnewline
\midrule
\endhead
\tabularnewline
\bottomrule
\end{longtable}

It is possible to hypothesize a local structural rearrangement that
transforms the chalcogen bond into a hydrogen bond, and \emph{vice
versa} . In other words, an oscillation from chalcogen to hydrogen bond.
This would optimize the bonding requirements and decrease the entropic
cost of protein folding.

It is nevertheless important to observe that the statistical analyses
presented in this manuscript cannot provide a quantitative estimation of
the energy associated with chalcogen and hydrogen bonds. There are in
fact several limitations. For example, although the position of the
hydrogen atoms is of crucial importance for defining hydrogen bonds, it
is usually unknown in protein crystal structures, especially for acidic
and rotatable hydrogen atoms. Moreover, without the hydrogen position,
one cannot identify chalcogen bonds that might be formed along the
extension of the H-S covalent bond. Moreover, chalcogen and hydrogen
bonds involving aromatic rings were not considered in the present study,
for the sake of simplicity, though they might participate in protein
structure stabilization, by forming both chalcogen and hydrogen bonds.

Further analyses, focused on X-ray and neutron protein crystal
structures at extremely high resolution might provide additional
information as well as analyses of protein structures determined with
alternative methods -- e.g. cryo-electron crystallography and nuclear
magnetic resonance in solution.

\section*{Materials and Methods}

{\label{materials-and-methods}}

\subsection*{Data selection}

{\label{data-selection}}

All data were extracted from the enormous amount of information
available in the Protein Data Bank {[}18{]}{[}19{]}. Only X-ray crystal
structures determined in the 80-120 K temperature range and refined at a
resolution of at least 2.0 \selectlanguage{ngerman}Å were retained. This resulted in about
66,500 entries of the Protein Data Bank.

Then two strategies were followed to extract non-redundant sets of data.

On the one hand, the pairwise sequence redundancy was reduced with
CD-HIT -- maximal percentage of sequence identity of 40\% {[}20{]} --
and the attention was limited to chains containing at least 50 amino
acids. This resulted in the \emph{Single} dataset containing about
14,000 protein chains.

On the other hand, the RaSPDB procedure was applied {[}21{]}. It
consists in creating several subsets of the Protein Data Bank. Each
subset must be large enough to be representative of the Protein Data
Bank and small enough to avoid internal redundancy. Nine non-overlapping
subsets, each containing about 7,000 protein chains made by more than 50
amino acids, were assembled, and all statistical analyses were performed
on each of them and then averaged. This procedure allows one to use a
much larger fraction of Protein Data Bank and to estimate the standard
errors of each estimate. This results in the nine
subsets\emph{raspdb\_X} (X=1-9).

\subsection*{Chalcogen bond detection}

{\label{chalcogen-bond-detection}}

In previous studies of chalcogen bonds formed by selenomethionine, the
position of the nucleophile relative to the selenium atom was described
by means of spherical coordinates {[}7{]}{[}11{]}, which require the
atomic positions of the C-Se-C triatomic fragment of the
selenomethionine side-chain. An analogous approach is impossible here,
where the attention is focused on the C-S-H triatomic fragment of
cysteine, given that the coordinates of this hydrogen atom are usually
unknown, since acidic and rotatable hydrogen atoms are often undetected,
even at very high crystallographic resolution or in neutron diffraction
studies.

In principle, it is possible to compute the position of these hydrogen
atoms by optimizing their interactions with atoms close by {[}22{]}.
This means by optimizing their hydrogen bonds {[}23{]}. Here it is
preferable to avoid the computation of the position of these hydrogen
atoms, since this would inevitably bias the analysis of chalcogen bonds.

As a consequence, a S-Nu chalcogen bond was simply defined as a contact
shorter than 3.4 Å (when Nu is an oxygen atom) or than 3.7 Å (when Nu is
a sulfur atom) and colinear or nearly colinear with the C-S bond, which
means that the angle \selectlanguage{greek}α \selectlanguage{english}= 180\selectlanguage{ngerman}°-(C\selectlanguage{greek}\textsubscript{β}-\selectlanguage{english}S\selectlanguage{greek}\textsubscript{γ}-\selectlanguage{english}Nu)
must be narrower than 25\selectlanguage{ngerman}° -- note that this threshold is larger than
20°, the value used in chemistry and material science, since it is
necessary to consider the lower accuracy of macromolecular crystal
structures.

Care was taken to remove from the chalcogen bonds' list the disulfide
bonds and the short sulfur-sulfur contacts that may be observed for
radiation damaged disulfide bonds {[}24{]}{[}25{]}. Analogously, short
sulfur-sulfur contacts resulting from the interactions of the sulfur
atoms with the same heteroatom -- typically a metal cation -- were
removed from the chalcogen bonds' list.

\subsection*{Hydrogen bond detection}

{\label{hydrogen-bond-detection}}

Potential hydrogen bonds that involve cysteine were identified with
HBPLUS {[}26{]} and filtered according to the following criteria
{[}27{]}{[}28{]}: S-A \textless{} 4.3 Å and S-A-AA \textgreater{} 90°
when the cysteine is a hydrogen donor; and D-S \textless{} 4.1 Å when
the cysteine is a hydrogen acceptor. Additional stereochemical criteria
that can be used to identify hydrogen bonds and that require the
knowledge of the position of the hydrogen atoms were disregarded, since
the hydrogen atom position is generally unknown.

\subsection*{Miscellaneous}

{\label{miscellaneous}}

Solvent accessible surface areas were computed with NACCESS {[}29{]} and
secondary structure assignments were performed with Stride {[}30{]}.

\section*{Author Contributions}

{\label{author-contributions}}

OC designed the procedures, executed all computations, and wrote the
manuscript.

\section*{Acknowledgements}

{\label{acknowledgements}}

Kristina Djinovi\selectlanguage{polish}ć \selectlanguage{english}is gratefully acknowledged for her kind hospitality
and prof. A. Stradella for his constant support.

\section*{References}

{\label{references}}

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\section*{Figure legends}

{\label{figure-legends}}

\textbf{Figure 1} . Scheme of the chalcogen and hydrogen bonds that may
involve the cysteine side-chain. \emph{Nu} indicates a
nucleophile,\emph{AA-A} a hydrogen acceptor, and \emph{D-H} a hydrogen
donor.

\textbf{Figure 2} . Example of chalcogen and hydrogen bonds formed by
the same cysteine thiol group (data from the file 1e9a of the Protein
Data Bank).

\section*{Supporting Information}

{\label{supporting-information}}

\textbf{Table S1}. Nucleophilic atom types that form chalcogen bonds
with the cysteine SG atom in the single dataset and in the nine subsets
of the Protein Data Bank generated with the RaSPDB procedure. The
average values (standard errors in parentheses) are computed on these
nine subsets.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
Atom & Dataset & Dataset & Dataset & Dataset & Dataset & Dataset &
Dataset & Dataset & Dataset & Dataset & Dataset\tabularnewline
& Single & raspdb\_1 & raspdb\_2 & raspdb\_3 & raspdb\_4 & raspdb\_5 &
raspdb\_6 & raspdb\_7 & raspdb\_8 & raspdb\_9 &
Average-1-9\tabularnewline
main-chain-O & 69.9 & 65.1 & 64.3 & 63.5 & 63.6 & 63.6 & 65.6 & 67.1 &
60.9 & 62.2 & 64.0(0.6)\tabularnewline
Ser-O & 1.0 & 0.0 & 0.0 & 1.1 & 0.6 & 0.9 & 1.2 & 1.7 & 1.4 & 1.2 &
0.9(0.2)\tabularnewline
Thr-O & 0.6 & 0.6 & 0.6 & 0.3 & 0.0 & 1.2 & 1.5 & 0.6 & 1.1 & 0.6 &
0.7(0.2)\tabularnewline
Tyr-O & 1.9 & 1.6 & 1.7 & 2.2 & 2.2 & 2.6 & 1.2 & 1.7 & 2.0 & 3.2 &
2.0(0.2)\tabularnewline
Asn-OD1 & 3.0 & 3.1 & 3.5 & 1.9 & 3.6 & 2.9 & 3.2 & 2.5 & 3.4 & 4.0 &
3.1(0.2)\tabularnewline
Gln-OE1 & 1.4 & 0.9 & 1.2 & 1.7 & 0.6 & 1.2 & 1.8 & 1.4 & 2.0 & 1.4 &
1.3(0.1)\tabularnewline
Asp-OD1-OD2 & 5.3 & 8.4 & 8.9 & 7.8 & 10.2 & 8.4 & 8.2 & 7.0 & 10.6 &
9.5 & 8.8(0.4)\tabularnewline
Gln-OE1-OE2 & 4.8 & 4.0 & 4.6 & 6.1 & 3.6 & 3.8 & 4.7 & 4.2 & 4.0 & 3.7
& 4.3(0.3)\tabularnewline
Cys-SG & 4.2 & 5.3 & 4.9 & 3.1 & 5.5 & 4.3 & 3.8 & 4.5 & 3.7 & 5.2 &
4.5(0.3)\tabularnewline
Met-SD & 7.1 & 9.3 & 9.2 & 10.0 & 8.8 & 9.2 & 8.2 & 7.5 & 9.5 & 7.8 &
8.9(0.3)\tabularnewline
HOH-O & 0.8 & 1.6 & 1.2 & 2.2 & 1.4 & 2.0 & 0.6 & 1.9 & 1.1 & 1.2 &
1.5(0.2)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S2}. Percentages of cases in which the Cys side-chain
behaves as a hydrogen-donor and as a hydrogen acceptor in the Single
dataset and in the nine subsets of the Protein Data Bank generated with
the RaSPDB procedure; the average values are computed on these nine
subsets (estimated standard errors in parentheses).\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Dataset & donor & acceptor\tabularnewline
Single & 69.2 & 30.8\tabularnewline
raspdb\_1 & 67.2 & 32.8\tabularnewline
raspdb\_2 & 67.1 & 32.9\tabularnewline
raspdb\_3 & 65.8 & 34.2\tabularnewline
raspdb\_4 & 66.1 & 33.9\tabularnewline
raspdb\_5 & 67.7 & 32.3\tabularnewline
raspdb\_6 & 66.9 & 33.1\tabularnewline
raspdb\_7 & 68.0 & 32.0\tabularnewline
raspdb\_8 & 66.9 & 33.1\tabularnewline
raspdb\_9 & 67.3 & 32.7\tabularnewline
Average-1-9 & 67.0(0.2) & 33.0(0.2)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S3}. Percentages of observation of various
hydrogen-acceptors from cysteine in the Single dataset and in the nine
subsets of the Protein Data Bank generated with the RaSPDB procedure.
The average values (with estimated standard errors in parentheses) are
computed on these nine subsets.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
Atom & Dataset & Dataset & Dataset & Dataset & Dataset & Dataset &
Dataset & Dataset & Dataset & Dataset & Dataset\tabularnewline
& Single & raspb\_1 & raspb\_2 & raspb\_3 & raspb\_4 & raspb\_5 &
raspb\_6 & raspb\_7 & raspb\_8 & raspb\_9 & Average-1-9\tabularnewline
main-chain-O & 79.7 & 80.9 & 80.5 & 79.2 & 80.3 & 81.0 & 79.9 & 80.7 &
79.7 & 80.3 & 80.27(0.20)\tabularnewline
Ser-OG & 3.0 & 2.7 & 2.9 & 3.2 & 3.0 & 2.7 & 3.0 & 2.9 & 3.1 & 3.1 &
2.94(0.06)\tabularnewline
Thr-OG1 & 2.9 & 2.8 & 2.8 & 2.8 & 2.7 & 3.1 & 2.7 & 2.7 & 2.9 & 2.8 &
2.80(0.04)\tabularnewline
Tyr-OH & 1.3 & 1.2 & 1.2 & 1.2 & 1.2 & 1.0 & 1.0 & 1.1 & 1.1 & 1.2 &
1.13(0.03)\tabularnewline
Asn-OD1 & 1.5 & 1.9 & 2.0 & 2.1 & 2.1 & 2.1 & 2.2 & 2.0 & 2.3 & 1.9 &
2.06(0.05)\tabularnewline
Gln-OE1 & 0.9 & 0.8 & 0.9 & 0.7 & 0.9 & 0.8 & 0.8 & 0.9 & 0.9 & 0.8 &
0.85(0.02)\tabularnewline
Asp-OD1-OD2 & 2.4 & 2.1 & 2.0 & 2.1 & 2.2 & 2.2 & 2.1 & 2.0 & 2.1 & 2.0
& 2.10(0.02)\tabularnewline
Glu-OE1-OE2 & 1.9 & 1.7 & 1.6 & 1.7 & 1.4 & 1.4 & 1.4 & 1.7 & 1.6 & 1.6
& 1.57(0.04)\tabularnewline
Cys-SG & 6.1 & 5.7 & 5.8 & 6.7 & 6.0 & 5.5 & 6.5 & 5.9 & 6.2 & 6.1 &
6.04(0.13)\tabularnewline
Met-SD & 0.3 & 0.3 & 0.2 & 0.3 & 0.2 & 0.2 & 0.3 & 0.3 & 0.2 & 0.2 &
0.25(0.01)\tabularnewline
HOH-O & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 &
0.00(0.00)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S4}. Percentages of observation of various hydrogen-donors
to cysteine in the Single dataset and in the nine subsets of the Protein
Data Bank generated with the RaSPDB procedure. The average values (with
estimated standard errors in parentheses) are computed on these nine
subsets.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
Atom & Dataset & Dataset & Dataset & Dataset & Dataset & Dataset &
Dataset & Dataset & Dataset & Dataset & Dataset\tabularnewline
& Single & raspdb\_1 & raspdb\_2 & raspdb\_3 & raspdb\_4 & raspdb\_5 &
raspdb\_6 & raspdb\_7 & raspdb\_8 & raspdb\_9 &
Average-1-9\tabularnewline
main-chain-N & 39.7 & 39.6 & 39.5 & 40.4 & 42.2 & 39.5 & 38.8 & 38.1 &
38.6 & 38.6 & 39.47(0.42)\tabularnewline
Ser-OG & 3.0 & 3.0 & 3.3 & 3.6 & 3.4 & 3.2 & 3.4 & 3.4 & 3.0 & 3.1 &
3.27(0.07)\tabularnewline
Thr-OG1 & 2.5 & 2.4 & 2.6 & 2.3 & 2.2 & 2.7 & 2.8 & 2.4 & 2.5 & 2.5 &
2.48(0.06)\tabularnewline
Tyr-OH & 0.5 & 0.6 & 0.8 & 0.7 & 0.7 & 0.5 & 0.4 & 0.5 & 0.6 & 0.6 &
0.61(0.04)\tabularnewline
Asn-ND2 & 0.6 & 0.4 & 0.3 & 0.3 & 0.4 & 0.4 & 0.3 & 0.3 & 0.5 & 0.5 &
0.39(0.03)\tabularnewline
Gln-NE2 & 0.5 & 0.7 & 0.8 & 0.7 & 0.4 & 0.7 & 0.6 & 0.6 & 0.6 & 0.6 &
0.63(0.03)\tabularnewline
His-ND1-NE2 & 0.6 & 0.6 & 0.6 & 0.4 & 0.6 & 0.5 & 0.6 & 0.6 & 0.5 & 0.5
& 0.53(0.03)\tabularnewline
Trp-NE1 & 0.2 & 0.5 & 0.6 & 0.5 & 0.5 & 0.5 & 0.5 & 0.5 & 0.5 & 0.5 &
0.51(0.01)\tabularnewline
Arg-NE-NH1-MH2 & 1.2 & 1.2 & 1.2 & 1.1 & 1.1 & 1.1 & 1.3 & 1.3 & 1.0 &
1.0 & 1.17(0.04)\tabularnewline
Lys-NZ & 14.3 & 12.2 & 12.8 & 13.7 & 12.3 & 12.5 & 14.0 & 13.4 & 13.6 &
13.8 & 13.15(0.23)\tabularnewline
Cys-SG & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 & 0.0 &
0.00(0.00)\tabularnewline
HOH-O & 36.9 & 38.8 & 37.6 & 36.2 & 36.1 & 38.3 & 37.3 & 38.9 & 38.5 &
38.4 & 37.79(0.36)\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-01/figure-01}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-02/figure-02}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

