The protein sequence and the natural variants of Mn-SOD were retrieved
from the UniProt database 
The UniProt ID is P04179, and currently, there are natural variants described
at six positions. The positions, the substitutions and their references in
UniProt are shown in .
Summary of identified SOD2 variants.
Non-synonymous SNP Analysis
The Mn-SOD variants were subjected to a variety of in silico
SNP analyses. The results of the non-synonymous SNP analyses are shown in .
Predictions of the effect of the missense variations on SOD2 protein
The SNPeffect workflow evaluates aggregation tendency (TANGO), amyloid
propensity (WALTZ), chaperone binding tendency (LIMBO) and protein stability
(FoldX). The natural variant E66V slightly enhances the protein stability,
in contrast with the G76R variant, which reduces the protein stability. The
I82T variant decreases the chaperone binding tendency, and the R156W variant
slightly reduces the protein stability.
According to PhD-SNP, variants S10I, A16V, G76R and I82T are neutral, whereas
variants E66V and R156W cause disease.
The PMUT analysis indicates that the natural variants S10I, E66V and I82T
are neutral and that A16V, G76R and R156W are pathological.
The PolyPhen-2 results show that, of the six variants, only E66V may cause
damage and that all of the others are benign.
According to SIFT (Sorting Intolerant from Tolerant), tolerance was predicted
for the natural variants S10I, A16V, E66V and G76R. I82T and R156W were predicted
to affect protein function. The SNAP analysis indicates that variants S10I,
G76R and I82T are non-neutral and that A16V, E66V and R156W are neutral.
According to SNPs&GO, variants S10I, G76R, I82T and R156W cause disease,
and A16V and E66V are neutral.
The nsSNPAnalyzer results demonstrate that variants S10I and A16V are unknown
and variants E66V and G76R cause disease. In contrast, I82T and R156W are
The SNP analysis, shown in , indicates that none of the natural variants have only positive results.
For each single mutation, at least one algorithm indicates a harmful effect
on the protein. This result demonstrates the importance of using different
algorithms because each algorithm uses different parameters to evaluate the
effects of natural variants.
Comparative and ab initio Modelling
The natural variants were substituted into the wild-type sequence for comparative
modelling. These sequences were submitted to the MHOLline workflow 
. The theoretical models
generated using MHOLline are presented in .
Superimposed native structures (green) and mutant structures (blue)
of the SOD2 produced using comparative modelling.
two chains of SOD2 (PDB ID: 1LUV), four mutations (the ones that are not in
the signal peptide) and the binding site for manganese. This figure indicates
that 3 of the variants localise in the interaction surfaces of chains A and
B. This localisation may adversely influence dimer formation, especially the
I58T mutation, which affects the stability of the tetrameric (dimer-dimer)
3D structure of human SOD2 with four missense mutation sites.
An alignment between the native and mutant structures was performed using
Parameters such as the TM-score and root mean square deviation (RMSD) were
used to analyse the topology and structural similarity of the models. TM-score
was used to assess the topological similarity of two protein structures, while
RMSD was the measure of the average distance between the backbones of the
superimposed proteins 
The RMSD values for the modelled mutants were significant for pathogenicity
for all missense mutations ( and ).
RMSD values greater than 0.15 were considered significant structural perturbations
that could have functional implications for the protein 
Structure alignment comparing mutant models and wild-type SOD2 models.
To analyse the three-dimensional effects of the S10I and A16V mutations,
which are located in the signal peptide, ab initio
was necessary because the signalling sequence cannot be resolved experimentally.
The I-Tasser server 
was utilised for the ab initio
modelling. As shown in and , the structural alignment of the ab
mutant models and the ab initio
reveals that the S10I and A16V mutations exhibited high RMSD values and disrupted
the alpha helix in the signal peptide.
Superimposed native structures (green) and mutant structures (blue)
of the SOD2 produced using ab initio modelling.
Structure alignment of ab initio SOD2 mutant models
with the ab initio wild-type model.
Structural Phylogenetic Analysis
The ConSurf 
results are based on the concept of identify functional regions in proteins,
taking into account by considering the evolutionary relationships among their
sequence homologues. An advantage of ConSurf over other methods is the accurate
computation of the evolutionary rate using either an empirical Bayesian method
or a maximum likelihood method. Thus, ConSurf can correctly discriminate between
the conservation caused by a short evolutionary time and genuine sequence
conservation. The surface residues with the most variation are depicted in
blue, and the conserved residues are depicted in purple in the protein structures
(). Our findings
revealed that human SOD2 is highly conserved (). The sequence alignment of the SOD2 from various species () reveals that residues
E66 and G76 are conserved, whereas I82 and R156 are variable.
Conservation profile of the Mn-SOD (PDB ID: 1LUV) using ConSurf conservational
Multiple protein sequence alignment using ConSurf shows evolutionary
conservation of amino acid residues.
The conservation analysis of ConSurf used the evolutionary conservation
scores of the residues to identify functional regions from proteins with known
three-dimensional structures. The degree of conservation of the amino acid
sites among the nine homologues with similar sequences () was estimated. The conservation grades
were projected onto the molecular surface of the proteins to reveal the patches
of highly conserved residues that are often important for biological function.
Mutations E66 and G76 are conserved, whereas mutations I82 and R156 are variable.
Generally, residues that are implicated in biological processes, such as those
located in active sites, involved in protein-protein or protein-ligand interactions,
or implicated in protein structure and folding stability, are subject to greater
selective pressure and are usually more conserved than other residues.
The SOD2 database currently contains all of the natural variants listed
in UniProt. For each SNP, we provide the predictions of functional effects,
indicated as Disease/Pathological or Neutral/Tolerated, from SNPeffect, PolyPhen-2,
PhD-SNP, PMUT, SIFT, SNAP, SNPs&GO and nsSNPAnalyzer.
The database interface () allows users to search for a mutation by its non-synonymous SNP.
Screenshot of the SOD2 Database web interface for structural modelling
and comparative analysis.
The database is curated by humans and will be updated as new natural variants
The SOD2 database allows a user to quickly retrieve and rapidly analyse
the predicted effects of protein variants. In addition to predicting the effects
of variants, an alignment of the wild-type and mutant structures can be visualised
using the database.
The major feature that distinguishes the SOD2 database from other databases
is that this database can use predictions from several algorithms for all
of the known natural variants of Mn-SOD. Furthermore, the user has access
to an alignment of the wild type and mutant structures and can thus visualise
the damage that a SNP can cause. Our ultimate goal is to turn the database
into a toolbox for researchers studying this protein. The in silico
analysis of Mn-SOD in this database will help in the design and prioritisation
of further experimental research.