**2. Validation and refinement**

Homology models are theoretical-computational approximations of the real protein structures, and therefore require validation and sometimes refinement and optimization. A very popular validation tool is the Ramachandran plot (**Figure 4**), which analyzes the stereochemical quality of protein structures.

The Ramachandran graph analyzes the conformations of the *phi* and *psi* angles of the peptide bonds, placing them in regions. Residues outside the permitted regions (outliers) are those that are in unfavorable configurations due to the collision between the atoms (steric shock). It preconizes that a good model should have at least 90% of its waste in favorable and permitted regions [5].

Other validation tools are energy assessments, both local and global ones. A tool for global assessment of the quality of a model is the server PROSA-web - Protein Structure Analysis (https://prosa.services.came.sbg.ac.at/prosa.php) [6, 7], which compares the energy of a structure with a database of proteins of equivalent size, solved experimentally, through the Z-score (**Figure 5**).

For local quality analysis, the application of the VERIFY3D server (https://servicesn.mbi.ucla.edu/Verify3D) is very useful. In this type of analysis it is possible to check the local quality, that is, for each residue of the model (**Figure 6**). With this, it is possible to identify specific regions of low quality for further adjustments.

For the models refinement, two techniques are particularly interesting: energy minimization and classical (atomistic) molecular dynamics. Energy minimization, also called optimization of geometry, aims to find a set of atomic coordinates of the structure that avoid bad contacts and reduce the potential energy of the system. There are some free servers available for energy minimization application in theoretical models, like YASARA [8] (http://www.yasara.org/minimizationserver.htm) and CHIRON [9] (https://dokhlab.med.psu.edu/chiron/). Molecular dynamics are extremely efficient for validating and refining theoretical models. This technique is based on the principles of Classical Mechanics and describes the atomic movements

#### **Figure 4.**

*Ramachandran graph for SARS-CoV-2 NSP9 replicase (PDB ID: 6w4b). In red, more favorable regions. In yellow and beige, regions allowed. In white, forbidden regions. Source: Authors data.*

**7**

**Figure 5.**

**Figure 6.**

*Source: Authors data.*

*resonance (dark blue). Source: Authors data.*

of a system through the integration of Newtonian equations of motion. Thus, a molecular dynamics simulation of 5–10 nanoseconds is one of the most effective techniques for optimization and validation of models by homology. For performing molecular dynamics calculations, software such as GROMACS [10] and NAMD [11] are useful. Once optimized and validated, the theoretical model can be used for several purposes, and can also be made available in public repositories, such as the PMDB - Protein Model DataBase (http://srv00.recas.ba.infn.it/PMDB/) and the

*Local ERRAT quality graph of a stretch from the NS5 enzyme from Zika virus. In blue, the average scores, in green, the raw scores. 93.93% of the residues have averaged 3D-1D score > = 0.2 (80% indicates good structures).* 

*Comparative graph of the Z-score energy. The black dot represents the position of the analyzed protein compared to equivalent size structures obtained by x-ray crystallography (light blue) and nuclear magnetic* 

SWISS-MODEL repository (https://swissmodel.expasy.org/repository).

*Introductory Chapter: Homology Modeling DOI: http://dx.doi.org/10.5772/intechopen.95446* *Introductory Chapter: Homology Modeling DOI: http://dx.doi.org/10.5772/intechopen.95446*

#### **Figure 5.**

*Homology Molecular Modeling - Perspectives and Applications*

which analyzes the stereochemical quality of protein structures.

at least 90% of its waste in favorable and permitted regions [5].

solved experimentally, through the Z-score (**Figure 5**).

Homology models are theoretical-computational approximations of the real protein structures, and therefore require validation and sometimes refinement and optimization. A very popular validation tool is the Ramachandran plot (**Figure 4**),

The Ramachandran graph analyzes the conformations of the *phi* and *psi* angles

Other validation tools are energy assessments, both local and global ones. A tool for global assessment of the quality of a model is the server PROSA-web - Protein Structure Analysis (https://prosa.services.came.sbg.ac.at/prosa.php) [6, 7], which compares the energy of a structure with a database of proteins of equivalent size,

For local quality analysis, the application of the VERIFY3D server (https://servicesn.mbi.ucla.edu/Verify3D) is very useful. In this type of analysis it is possible to check the local quality, that is, for each residue of the model (**Figure 6**). With this, it

For the models refinement, two techniques are particularly interesting: energy minimization and classical (atomistic) molecular dynamics. Energy minimization, also called optimization of geometry, aims to find a set of atomic coordinates of the structure that avoid bad contacts and reduce the potential energy of the system. There are some free servers available for energy minimization application in theoretical models, like YASARA [8] (http://www.yasara.org/minimizationserver.htm) and CHIRON [9] (https://dokhlab.med.psu.edu/chiron/). Molecular dynamics are extremely efficient for validating and refining theoretical models. This technique is based on the principles of Classical Mechanics and describes the atomic movements

is possible to identify specific regions of low quality for further adjustments.

*Ramachandran graph for SARS-CoV-2 NSP9 replicase (PDB ID: 6w4b). In red, more favorable regions.* 

*In yellow and beige, regions allowed. In white, forbidden regions. Source: Authors data.*

of the peptide bonds, placing them in regions. Residues outside the permitted regions (outliers) are those that are in unfavorable configurations due to the collision between the atoms (steric shock). It preconizes that a good model should have

**2. Validation and refinement**

**6**

**Figure 4.**

*Comparative graph of the Z-score energy. The black dot represents the position of the analyzed protein compared to equivalent size structures obtained by x-ray crystallography (light blue) and nuclear magnetic resonance (dark blue). Source: Authors data.*

#### **Figure 6.**

*Local ERRAT quality graph of a stretch from the NS5 enzyme from Zika virus. In blue, the average scores, in green, the raw scores. 93.93% of the residues have averaged 3D-1D score > = 0.2 (80% indicates good structures). Source: Authors data.*

of a system through the integration of Newtonian equations of motion. Thus, a molecular dynamics simulation of 5–10 nanoseconds is one of the most effective techniques for optimization and validation of models by homology. For performing molecular dynamics calculations, software such as GROMACS [10] and NAMD [11] are useful. Once optimized and validated, the theoretical model can be used for several purposes, and can also be made available in public repositories, such as the PMDB - Protein Model DataBase (http://srv00.recas.ba.infn.it/PMDB/) and the SWISS-MODEL repository (https://swissmodel.expasy.org/repository).
