Steps of Homology Modeling
The first step in protein structural modeling is to select appropriate structural templates. The template selection involves searching the Protein Data Bank (PDB) for homologous proteins with determined structures. The search can be performed using a heuristic pairwise alignment search program such as BLAST or FASTA. As a rule of thumb, a database protein should have at least 30% sequence identity with the query sequence to be selected as template. Occasionally, a 20% identity level can be used as threshold as long as the identity of the sequence pair falls within the “safe zone”.
Once the structure with the highest sequence similarity is identified as a template, the full-length sequences of the template and target proteins need to be realigned using refined alignment algorithms to obtain optimal alignment. Errors made in the alignment step cannot be corrected in the following modeling steps. Therefore, the best possible multiple alignment algorithms, such as Praline and T-Coffee, should be used for this purpose.
Backbone Model Building
Once optimal alignment is achieved, residues in the aligned regions of the target protein can assume a similar structure as the template proteins, meaning that the coordinates of the corresponding residues of the template proteins can be simply copied onto the target protein. If the two aligned residues are identical, coordinates of the side chain atoms are copied along with the main chain atoms. If the two residues differ, only the backbone atoms can be copied.
In the sequence alignment for modeling, there are often regions caused by insertions and deletions producing gaps in sequence alignment. The gaps cannot be directly modeled, creating “holes” in the model. Closing the gaps requires loop modeling, which is a very difficult problem in homology modeling and is also a major source of error. Loop modeling can be considered a mini–protein modeling problem by itself. Currently, there are two main techniques used to approach the problem: the database searching method and the ab initio method. The database method involves finding “spare parts” from known protein structures in a database that fit onto the two stem regions of the target protein. The stems are defined as the main chain atoms that precede and follow the loop to be modeled. The procedure begins by measuring the orientation and distance of the anchor regions in the stems and searching PDB for segments of the same length that also match the above endpoint conformation. The ab initio method generates many random loops and searches for the one that does not clash with nearby side chains and also has reasonably low energy and φ and ψ angles in the allowable regions in the Ramachandran plot.
Side Chain Refinement
Once main chain atoms are built, the positions of side chains that are not modeled must be determined. Modeling side chain geometry is very important in evaluating protein–ligand interactions at active sites and protein–protein interactions at the contact interface. A side chain can be built by searching every possible conformation at every torsion angle of the side chain to select the one that has the lowest interaction energy with neighboring atoms. A collection of preferred side chain conformations is a rotamer library in which the rotamers are ranked by their frequency of occurrence. Having a rotamer library reduces the computational time significantly because only a small number of favored torsion angles are examined. In prediction of side chain conformation, only the possible rotamers with the lowest interaction energy with nearby atoms are selected. After adding the most frequently occurring rotamers, the conformations have to be further optimized to minimize steric overlaps with the rest of the model structure. Most modeling packages incorporate the side chain refinement function.
Model Refinement Using Energy Function
In these loop modeling and side chain modeling steps, potential energy calculations are applied to improve the model. However, this does not guarantee that the entire raw homology model is free of structural irregularities such as unfavorable bond angles, bond lengths, or close atomic contacts. These kinds of structural irregularities can be corrected by applying the energy minimization procedure on the entire model, which moves the atoms in such a way that the overall conformation has the lowest energy potential. Key conserved residues and those involved in cofactor binding have to be restrained if necessary during the process. Another often used structure refinement procedure is molecular dynamic simulation.
The final homology model has to be evaluated to make sure that the structural features of the model are consistent with the physicochemical rules. This involves checking anomalies in φ–ψ angles, bond lengths, close contacts, and so on. Another way of checking the quality of a protein model is to implicitly take these stereochemical properties into account. By comparing the statistical parameters with the constructed model, the method reveals which regions of a sequence appear to be folded normally and which regions do not. If structural irregularities are found, the region is considered to have errors and has to be further refined.