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Computational Molecular Biology
4
nucleotide database (Acc. No. NC_013851). These
amino acid sequences were used separately to build
homology models by modeller by discovery studio.
These amino acid sequences were used to do BLAST
against Protein Data Bank to find suitable template for
homology modelling. The search result showed the
X-ray crystal structure from
Chlorobium Limicola F
Thiosulfatophilum
(PDB code: 2NNC A chain for
SoxY) and from
Paracoccus Pantotrophus
(PDB code:
2OXG Z chain for SoxZ). The sequences of SoxY and
SoxZ share 49% and 36% sequence identity
respectively with template sequences. Due to large
SoxB protein sequence of only C-terminal domain
(5'-nucleotidase, C-terminal domain) had been
modelled using discovery studio modeller. Pfam was
used to identify conserved domain of this protein. The
same was also verified by BLAST.The domain was
146 amino acid residue lengths. For SoxB domain the
best template was to be the x-ray crystal structure of
Sulfate Thiohydrolase Soxb from
Termus Thermophilus
(PDB code: 2WDC A chain for SoxB domain) with
sequence identity 44%. The root-mean-square
deviation (RMSD) is used to study the globular
protein conformation. The RMSD for each and
individual model protein structure was calculated by
superimposing separately on each of the crystal
templates about their main chain conformation (A
chain of 2NNC for SoxY, Z chain of 2OXG for SoxZ
and A chain of 2WDC for SoxB 5'-nucleotidase,
C-terminal
domain).
The RMSD for the
superimpositions were 0.255 Ǻ for SoxY, 1.117 Ǻ for
SoxZ and 0.477Ǻ for 5'-nucleotidase, C-terminal
domain SoxB.
The models of the proteins were then energy
minimized in two steps. In first step the steepest
decent technique was used and in the next step
conjugate gradient technique was used to minimize
the overall structure of the three models using the
Discovery studio software until the structures reached
the final RMS gradient of 0.0001. All energy
minimization were done using CHARRAM force field
and fixing backbone of proteins (Brooks et al., 1983).
3.2 Validation of models
The z score of each and individual model protein are
calculated using Prosa webserver. The Z-score showed
that predicted model structures were well inside the
native structure (Sippl, 1993). Saves server was used
to verify the main chain properties of the modelled
proteins. No considerable bad contacts or C
α
tetrahedron distortions were found. VERIFY3D was
used to check amino acid residue profile of the three
dimensional models (Eisenberg et al., 1997). The
stereo chemical qualities of the models and
Ramachandran plots were analysed using
PROCHECK web server (Laskowski et al., 1993).
In the Ramachandran plot no residues were found
to be in disallowed region (Ramachandran and
Sashisekharan, 1968).
3.3 Molecular docking simulations
In order to study the interactions between SoxYZ
complex protein and 5'-nucleotidase, C-terminal
domain SoxB and first the models of the SoxY and
SoxZ proteins were docked using the software
Cluspro 2.0 (Comeau et al., 2004). Cluspro 2.0 is a
fully automated web based program for
protein-protein docking (Comeau et al., 2004). The
docked structure of SoxY and SoxZ protein was again
docked with model structure of 5'-nucleotidase,
C-terminal domain of SoxB protein of
A.vino
using
Cluspro 2.0 protein-protein docking server. Using
advanced option of Cluspro 2.0 the unstructured
residues from receptor and ligand was removed. The
model 0 was chosen from different displayed model
structures because it had the best cluster size among
all the possible docked structures, was selected and
analysed. The complex structure was energy
minimized by fixing backbone of the proteins in
complex by the steepest descent technique using
CHARRAM force fields (Brooks et al., 1983).
3.4 Calculation of protein-protein interaction
To find out the interactions between the SoxY, SoxZ
and SoxB domain, Protein Interaction Calculator
(P.I.C) web server was used. P.I.C web server is an
online web server in which protein three dimensional
structures was provided to calculate various kinds of
interactions; such as disulphide bonds, hydrophobic
interactions,
ionic interactions,
hydrogen bonds,
aromatic-aromatic interactions, aromatic-sulphur interactions
and cation - π interactions within a protein or between
proteins in a complex (Tina et al., 2007).
Computational
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