Download Alignment between domain region and whole enzyme

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An in-silico analysis showing the interaction of FAD ligand with cytokinin
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dehydrogenase enzyme and with its domain part in rice
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ABSTRACT
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The cytokinin dehydrogenase enzyme plays an important role in the high grain
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production of rice. This enzyme interacts with the FAD ligand. This study shows
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the domain region of the enzyme which was identified by protparam tool,
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generated homology models using modeller9.12 tool, models were validated using
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errat, procheck saves server, prosa and anolea server. Then the active sites were
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predicted by using castP server. Finally the interaction was studied between the
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FAD and cytokinin dehydrogenase and with its domain part. The stronger
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interaction was found between the FAD and the domain of the cytokinin
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degydrogenase with the binding enegy of -10.91. ALA6, ARG1, AR536, TYR10,
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ASP55, ALA57, CYS38 of FAD is binding to the domain part of cytokinin
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degydrogenase in rice.
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INTRODUCTION
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Cytokinin dehydrogenase of Oryza sativa (japonica) plays an important role in
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high grain production. The cytokinin dehydrogenase helps in the oxidation of
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cytokinins. This protein belongs to the family of N(6)-substituted adenine
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derivatives, which is a plant hormone having isopentenyl group. This protein helps
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in modulateing the number of reproductive organs by regulating the cytokinin
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accumulation in inflorescence meristems. The CKX2 is also known as OsCKX2.
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The CKX2 is generally located on Os01g0197700. FAD or flavoprotein is the
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ligand of Cytokinin dehydrogenase 2 protein in O.sativa.CKX2 is located on
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chromosome number 1. CKX2 has 5137 basepairs. Cytokinin positively regulates
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the growth of shoot apical meristem that helps in more seed production. The Gn1a
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gene of rice encodes cytokinin oxidase as a major quantitative trait locus. zinc
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finger transcription factor directly regulates the OsCKX2 expression in the
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reproductive meristem [1]. Reduced expression of OsCKX2 causes cytokinin
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accumulation in inflorescence meristems leading to the increase in number of
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reproductive organs which results more grain production [2]. CKX helps for high
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grain production in wheat, maize, barely, sorghum, foxtail millet [3]. Cytokinin
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dehydrogenase plays an important role in the formation of crown root system in
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rice, which helps in high grain production [4]. The main objective of this study is
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to find out the best interaction among the ligand FAD and enzyme. Here the
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interaction is studied by considering the whole amino acid sequence and then only
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the domain part. Domain is the function region of a protein or domain. So
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generally the binding is more stronger at domain part. This study involves retrieval
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of amino acid sequence, predicting the domain region of enzyme, finding the
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common residues among domain part and the whole sequence, generating
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homologous models, validating and evaluating models, predicting the active site,
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interaction of FAD with the enzyme.
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MATERIAL AND METHODS
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Sequence retrieval
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The amino acid sequences of cytokinin dehydrogenase was retrieved from uniprot
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in fasta format.
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Finding the domain part
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The sequence of domain in the protein was retrieved by using protparam tool.
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However the domain region can be found from CDD (conserved domain database)
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too.
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Alignment between whole protein sequence and domain
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Multiple sequenece alignment is generally done to find out the conserved residues
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among multiple sequences[5]. From the alignment we found the regions of
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common residues among the domain part and whole sequence depending upon
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which we could analyse the stronger binding region where ligand (FAD) is
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binding. The alignment result is shown in fig.1.
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Predicting the structures of domain and whole protein
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As the NMR crystallographic structure of enzyme is not available at PDB, so the
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tertiary structure was generated on the basis of homology modeling concept [6]-
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[8].The models for whole protein and domain parts were generated using
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modeller9.12 tool. The align2d.py, model-single.py, evaluate-model.py files were
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run on the python script by setting the template, target and the number of models to
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be generated. In this study 10 models were generated for both domain part and for
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whole enzyme sequence. The best model was selected on the basis of lowest
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DOPE score. The final generated models were visualized by using visualizing tools
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i.e, pymol, discovery studio and swiss-pdb viewer. Multiple visualiser tools were
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used to understand the structures more properly (table1,table2). The final models
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were shown in fig.2 and fig.3.
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Model validation and optimization
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Expasy server provides many online tools with the help of which the generated
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protein models can be evaluated and validated [9]. The generated models were then
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validated by using Errat, Verify3d, Procheck saves server. The backbone
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confirmation of the protein was studied by using procheck saves server from where
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the residues lying in favoured region, allowed region and outlier regions can be
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found. When more residues found in favoured region and less residues found in
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outlier region, then it represents that protein is stable for further study (table3).
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Then the final Z-score of the models were obtained from prosa server, which
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showed the overall model quality (table.4b). The models were then submitted to
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Anolea server which gave the QMEAN score and Z-score (table.4a).
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Active site prediction
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The active site or binding site is the region where the ligand binds to receptor. In
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this study the active site was predicted by using castp server [10], [11]. The active
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sites of both enzyme and domain are shown in fig.4 and fig.5. The active sites
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shows the region where the ligand can bind or not. The binding energy dependent
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upon the type of residue to which it is binding i.e, hydrophobic or hydrophilic or
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polar or no-polar etc. the residues found at active sites are given in fig.6 and fig.7
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respectively.
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Protein-ligand interaction
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The interaction of ligand (FAD) was checked with whole enzyme and only with
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the domain parts separately to find out the regions where the lignad binds properly.
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The interaction was studied by using autodock tool [12], [13]. The pdb files of both
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enzyme, domain, ligand were uploaded one after another by setting the docking
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parameters. The autodock and autogrid files were generally run on python script
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after which the interactions found by analyzing the dock.dlg files. Less the binding
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energy more stronger is the interaction. Howerevr the binding energy is dependent
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on the length of amino acids. The interaction result is shown in fig.8 and fig.9
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respectively.
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RESULT
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Retrieved sequence and finding the domain region
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The amino acid sequence of enzyme was retrieved from uniprot with the uniprotID
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of Q4ADV8. The protein contains 565 number of amino acids. The domain region
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is found from 74-225. The information about the domain region was found from
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both uniprot and from protparam output.
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Alignment between domain region and whole enzyme
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The domain region was extracted from the whole protein sequence and subjected
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to clustalw2 for multiple sequence alignment. From this study the conserved
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residues among domain region and whole enzyme sequence were obtained. The
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match regions were denoted with *, mis-match regions with . and the gaps were
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denoted with -. Here the fig.1 shows the common residues.
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Homology modelling analysis
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The homologous models for both enzyme and domains were generated. The
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helices are denoted red colours, helices with yellow colours and the loops are
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denoted with green colours respectively. The VDW radius, beta factor, mass of
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structures, stability of the objects were found from yasara tool. Then total number
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of atoms, formal charge sum, molecular surface area and solvent accessible surface
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area were found from pymol tool.
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Model validation and optimization
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The backbone confirmations of the structures were generated from rampage server.
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91.8% of the residues lie in most favoured region whereas 3.3% of the residues lie
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in outlier region. The QMEAN score and Z-scores were found from both anolea
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server and from prosa server.
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Active site analysis result
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The active sites were found from castp server. The balls with green colour shows
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the binding regions of whole protein and the domain region. Many pockets were
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found, but out of them the first volume was selected. The pockets of domain were
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mainly ALA, ARG, CYS, HIS, GLY, VAL, SER, TYU, LEU, PHE, ILE, THR,
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PRO and similarly the pockets of whole enzyme were PHE, ILE, LEU, VAL, SER,
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HIS, GLY, CYS, ALA, MET, GLU, THR, ASP, TYR, ASN, GLN, PRO, TRP,
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GLN respectively.
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Protein-ligand interaction
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The pdb files of protein, domain and the ligand were submitted and by adding
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polar hydrogens to protein and domain the interaction was studied. The binding
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energy which was found between the interaction of whole protein-ligand (FAD)
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was -11.23, with -14.81 intermolecular energy, 3.58 torsion energy and 13.66
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internal energy. Similarly the interaction which was performed between the
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domain region and ligand (FAD) showed -10.91 binding energy, -14.49
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intermolecular energy, 3.58 torsion energy and 14.04 internal energy respectively.
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From the interaction it is found that the FAD is binding to ALA6, ARG1, AR536,
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TYR10, ASP55, ALA57, CYS38 at the domain part and in whole protein the
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domain is binding to ARG134, ASP128, SER131, TYR83, SER85, LEU135,
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ARG86, GLN136, LEU135, LEU47.
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DISCUSSION
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From the above study it is found that the model of whole enzyme and the domain
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are suitable for future analysis as most of the residues lie in favoured region. The
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main objective of this study was to find the stronger interaction of ligand (FAD)
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with the domain part of enzyme and with the whole sequence, which showed that
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the binding interaction is more stronger between the domain part and FAD than in
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comparision to the interaction between FAD and whole amino acid sequence.
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Angeles ER, Qian Q, Kitano H, Matsuoka M, Cytokinin oxidase regulates
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Gao S, Fang J, Xu F, Wang W, Sun X, Chu J, Cai B, Feng Y, Chu C,
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Fig.1 multiple sequence alignment between domain and whole enzyme
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Fig.2 Model generated for whole enzyme
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Fig.3 Model generated for domain
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Fig.4 Binding site of whole enzyme
Fig.5 Binding site of domain
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Fig.6 residues present at the active site of whole enzyme
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Fig.7 residues present at the active site of domain of the enzyme
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Fig.8 FAD binding to whole enzyme
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Fig.9 FAD binding to domain
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Table.1 information found after visualizing the structures using Pymol tool
Analysis
Protein structure
Domain structure
Atom count
4233
1311
Formal charge sum
-6.0
0.0
Molecular surface area
52371.758A0
16504.783 A0
Solvent accessible surface area
23178.145 A0
10648.261 A0
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Table.2 information found after visualizing the structures using Yasara tool
Analysis
Protein structure
Domain structure
VDW radius
85.337
34.261
Beta factor
109.7
111.3
Mass of object
55813.069g/mol
17316.863g/mol
Stability of the object
971.13kcal/mol
286.78kcal/mol
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Table.3 The backbone confirmation of models found from rampage server
Analysis
Protein structure Domain structure
Number of residues in favoured region
517 ( 91.8%)
159 ( 88.3%)
Number of residues in allowed region
34 ( 6.0%)
15 ( 8.3%)
Number of residues in outlier region
12 ( 2.1%)
6 ( 3.3%)
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Table.4 Model validation result
4a-
Result found from anolea server
Analysis
protein
Domain
QMEAN score
0.452
0.628
Z-score
-3.206
-1.483
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4b-
Result found from prosa server
Analysis
protein
Domain
Z-score
-8.32
-4.21
225
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