Homology modeling

Homology modeling

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Encyclopedia
Homology modeling, also known as comparative modeling of protein refers to constructing an atomic-resolution model of the "target" protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 from its amino acid sequence
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...

 and an experimental three-dimensional structure of a related homologous protein (the "template"). Homology modeling relies on the identification of one or more known protein structures likely to resemble the structure of the query sequence, and on the production of an alignment
Sequence alignment
In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Aligned sequences of nucleotide or amino acid residues are...

 that maps residues in the query sequence to residues in the template sequence. It has been shown that protein structures are more conserved than protein sequences amongst homologues, but sequences falling below a 20% sequence identity can have very different structure.

Evolutionarily related proteins have similar sequences and naturally occurring homologous proteins have similar protein structure.
It has been shown that three-dimensional protein structure is evolutionarily more conserved than expected due to sequence conservation.

The sequence alignment and template structure are then used to produce a structural model of the target. Because protein structures are more conserved than DNA sequences, detectable levels of sequence similarity usually imply significant structural similarity.

The quality of the homology model is dependent on the quality of the sequence alignment and template structure. The approach can be complicated by the presence of alignment gaps (commonly called indels) that indicate a structural region present in the target but not in the template, and by structure gaps in the template that arise from poor resolution in the experimental procedure (usually X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...

) used to solve the structure. Model quality declines with decreasing sequence identity; a typical model has ~1–2 Å
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

 root mean square deviation
Root mean square deviation
The root-mean-square deviation is the measure of the average distance between the atoms of superimposed proteins...

 between the matched Cα atoms at 70% sequence identity but only 2–4 Å
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

 agreement at 25% sequence identity. However, the errors are significantly higher in the loop regions, where the amino acid sequences of the target and template proteins may be completely different.

Regions of the model that were constructed without a template, usually by loop modeling
Loop modeling
Loop modeling is a problem in protein structure prediction requiring the prediction of the conformations of loop regions in proteins without the use of a structural template. The problem arises often in homology modeling, where the tertiary structure of an amino acid sequence is predicted based on...

, are generally much less accurate than the rest of the model. Errors in side chain
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...

 packing and position also increase with decreasing identity, and variations in these packing configurations have been suggested as a major reason for poor model quality at low identity. Taken together, these various atomic-position errors are significant and impede the use of homology models for purposes that require atomic-resolution data, such as drug design
Drug design
Drug design, also sometimes referred to as rational drug design or structure-based drug design, is the inventive process of finding new medications based on the knowledge of the biological target...

 and protein–protein interaction predictions; even the quaternary structure
Quaternary structure
In biochemistry, quaternary structure is the arrangement of multiple folded protein or coiling protein molecules in a multi-subunit complex.-Description and examples:...

 of a protein may be difficult to predict from homology models of its subunit(s). Nevertheless, homology models can be useful in reaching qualitative conclusions about the biochemistry of the query sequence, especially in formulating hypotheses about why certain residues are conserved, which may in turn lead to experiments to test those hypotheses. For example, the spatial arrangement of conserved residues may suggest whether a particular residue is conserved to stabilize the folding, to participate in binding some small molecule, or to foster association with another protein or nucleic acid.

Homology modeling can produce high-quality structural models when the target and template are closely related, which has inspired the formation of a structural genomics
Structural genomics
Structural genomics seeks to describe the 3-dimensional structure of every protein encoded by a given genome. This genome-based approach allows for a high-throughput method of structure determination by a combination of experimental and modeling approaches...

 consortium dedicated to the production of representative experimental structures for all classes of protein folds. The chief inaccuracies in homology modeling, which worsen with lower sequence identity, derive from errors in the initial sequence alignment and from improper template selection. Like other methods of structure prediction, current practice in homology modeling is assessed in a biannual large-scale experiment known as the Critical Assessment of Techniques for Protein Structure Prediction, or CASP
CASP
CASP, which stands for Critical Assessment of Techniques for Protein Structure Prediction, is a community-wide, worldwide experiment for protein structure prediction taking place every two years since 1994...

.

Motive


The method of homology modeling is based on the observation that protein tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

 is better conserved than amino acid sequence
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...

. Thus, even proteins that have diverged appreciably in sequence but still share detectable similarity will also share common structural properties, particularly the overall fold. Because it is difficult and time-consuming to obtain experimental structures from methods such as X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...

 and protein NMR for every protein of interest, homology modeling can provide useful structural models for generating hypotheses about a protein's function and directing further experimental work.

There are exceptions to the general rule that proteins sharing significant sequence identity will share a fold. For example, a judiciously chosen set of mutations of less than 50% of a protein can cause the protein to adopt a completely different fold. However, such a massive structural rearrangement is unlikely to occur in evolution
Evolution
Evolution is any change across successive generations in the heritable characteristics of biological populations. Evolutionary processes give rise to diversity at every level of biological organisation, including species, individual organisms and molecules such as DNA and proteins.Life on Earth...

, especially since the protein is usually under the constraint that it must fold
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

 properly and carry out its function in the cell. Consequently, the roughly folded structure of a protein (its "topology") is conserved longer than its amino-acid sequence and much longer than the corresponding DNA sequence; in other words, two proteins may share a similar fold even if their evolutionary relationship is so distant that it cannot be discerned reliably. For comparison, the function of a protein is conserved much less than the protein sequence, since relatively few changes in amino-acid sequence are required to take on a related function.

Steps in model production


The homology modeling procedure can be broken down into four sequential steps: template selection, target-template alignment, model construction, and model assessment. The first two steps are often essentially performed together, as the most common methods of identifying templates rely on the production of sequence alignments; however, these alignments may not be of sufficient quality because database search techniques prioritize speed over alignment quality. These processes can be performed iteratively to improve the quality of the final model, although quality assessments that are not dependent on the true target structure are still under development.

Optimizing the speed and accuracy of these steps for use in large-scale automated structure prediction is a key component of structural genomics initiatives, partly because the resulting volume of data will be too large to process manually and partly because the goal of structural genomics requires providing models of reasonable quality to researchers who are not themselves structure prediction experts.

Template selection and sequence alignment


The critical first step in homology modeling is the identification of the best template structure, if indeed any are available. The simplest method of template identification relies on serial pairwise sequence alignments aided by database search techniques such as FASTA
FASTA
FASTA is a DNA and protein sequence alignment software package first described by David J. Lipman and William R. Pearson in 1985. Its legacy is the FASTA format which is now ubiquitous in bioinformatics.- History :...

 and BLAST
BLAST
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences...

. More sensitive methods based on multiple sequence alignment
Multiple sequence alignment
A multiple sequence alignment is a sequence alignment of three or more biological sequences, generally protein, DNA, or RNA. In many cases, the input set of query sequences are assumed to have an evolutionary relationship by which they share a lineage and are descended from a common ancestor...

 – of which PSI-BLAST is the most common example – iteratively update their position-specific scoring matrix
Position-specific scoring matrix
A position weight matrix , also called position-specific weight matrix or position-specific scoring matrix , is a commonly used representation of motifs in biological sequences....

 to successively identify more distantly related homologs. This family of methods has been shown to produce a larger number of potential templates and to identify better templates for sequences that have only distant relationships to any solved structure. Protein threading , also known as fold recognition or 3D-1D alignment, can also be used as a search technique for identifying templates to be used in traditional homology modeling methods. When performing a BLAST search, a reliable first approach is to identify hits with a sufficiently low E-value, which are considered sufficiently close in evolution to make a reliable homology model. Other factors may tip the balance in marginal cases; for example, the template may have a function similar to that of the query sequence, or it may belong to a homologous operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...

. However, a template with a poor E-value should generally not be chosen, even if it is the only one available, since it may well have a wrong structure, leading to the production of a misguided model. A better approach is to submit the primary sequence to fold-recognition servers or, better still, consensus meta-servers which improve upon individual fold-recognition servers by identifying similarities (consensus) among independent predictions.

Often several candidate template structures are identified by these approaches. Although some methods can generate hybrid models from multiple templates, most methods rely on a single template. Therefore, choosing the best template from among the candidates is a key step, and can affect the final accuracy of the structure significantly. This choice is guided by several factors, such as the similarity of the query and template sequences, of their functions, and of the predicted query and observed template secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

s. Perhaps most importantly, the coverage of the aligned regions: the fraction of the query sequence structure that can be predicted from the template, and the plausibility of the resulting model. Thus, sometimes several homology models are produced for a single query sequence, with the most likely candidate chosen only in the final step.

It is possible to use the sequence alignment generated by the database search technique as the basis for the subsequent model production; however, more sophisticated approaches have also been explored. One proposal generates an ensemble of stochastic
Stochastic
Stochastic refers to systems whose behaviour is intrinsically non-deterministic. A stochastic process is one whose behavior is non-deterministic, in that a system's subsequent state is determined both by the process's predictable actions and by a random element. However, according to M. Kac and E...

ally defined pairwise alignments between the target sequence and a single identified template as a means of exploring "alignment space" in regions of sequence with low local similarity. "Profile-profile" alignments that first generate a sequence profile of the target and systematically compare it to the sequence profiles of solved structures; the coarse-graining inherent in the profile construction is thought to reduce noise introduced by sequence drift
Genetic drift
Genetic drift or allelic drift is the change in the frequency of a gene variant in a population due to random sampling.The alleles in the offspring are a sample of those in the parents, and chance has a role in determining whether a given individual survives and reproduces...

 in nonessential regions of the sequence.

Model generation


Given a template and an alignment, the information contained therein must be used to generate a three-dimensional structural model of the target, represented as a set of Cartesian coordinates for each atom in the protein. Three major classes of model generation methods have been proposed.

Fragment assembly


The original method of homology modeling relied on the assembly of a complete model from conserved structural fragments identified in closely related solved structures. For example, a modeling study of serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

s in mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...

s identified a sharp distinction between "core" structural regions conserved in all experimental structures in the class, and variable regions typically located in the loops where the majority of the sequence differences were localized. Thus unsolved proteins could be modeled by first constructing the conserved core and then substituting variable regions from other proteins in the set of solved structures. Current implementations of this method differ mainly in the way they deal with regions that are not conserved or that lack a template. The variable regions are often constructed with the help of fragment libraries
Protein fragment library
Protein backbone fragment libraries have been used successfully in a variety of structural biology applications, including homology modeling, de novo structure prediction, and structure determination...

.

Segment matching


The segment-matching method divides the target into a series of short segments, each of which is matched to its own template fitted from the Protein Data Bank
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....

. Thus, sequence alignment is done over segments rather than over the entire protein. Selection of the template for each segment is based on sequence similarity, comparisons of alpha carbon
Alpha carbon
The alpha carbon in organic chemistry refers to the first carbon that attaches to a functional group . By extension, the second carbon is the beta carbon, and so on....

 coordinates, and predicted steric conflicts arising from the van der Waals radii
Van der Waals radius
The van der Waals radius, r, of an atom is the radius of an imaginary hard sphere which can be used to model the atom for many purposes. It is named after Johannes Diderik van der Waals, winner of the 1910 Nobel Prize in Physics, as he was the first to recognise that atoms had a finite size and to...

 of the divergent atoms between target and template.

Satisfaction of spatial restraints


The most common current homology modeling method takes its inspiration from calculations required to construct a three-dimensional structure from data generated by NMR spectroscopy
NMR spectroscopy
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained...

. One or more target-template alignments are used to construct a set of geometrical criteria that are then converted to probability density function
Probability density function
In probability theory, a probability density function , or density of a continuous random variable is a function that describes the relative likelihood for this random variable to occur at a given point. The probability for the random variable to fall within a particular region is given by the...

s for each restraint. Restraints applied to the main protein internal coordinates – protein backbone distances and dihedral angle
Dihedral angle
In geometry, a dihedral or torsion angle is the angle between two planes.The dihedral angle of two planes can be seen by looking at the planes "edge on", i.e., along their line of intersection...

s – serve as the basis for a global optimization
Global optimization
Global optimization is a branch of applied mathematics and numerical analysis that deals with the optimization of a function or a set of functions to some criteria.- General :The most common form is the minimization of one real-valued function...

 procedure that originally used conjugate gradient energy minimization to iteratively refine the positions of all heavy atoms in the protein.

This method had been dramatically expanded to apply specifically to loop modeling, which can be extremely difficult due to the high flexibility of loops in proteins in aqueous solution. A more recent expansion applies the spatial-restraint model to electron density
Electron density
Electron density is the measure of the probability of an electron being present at a specific location.In molecules, regions of electron density are usually found around the atom, and its bonds...

 maps derived from cryoelectron microscopy studies, which provide low-resolution information that is not usually itself sufficient to generate atomic-resolution structural models. To address the problem of inaccuracies in initial target-template sequence alignment, an iterative procedure has also been introduced to refine the alignment on the basis of the initial structural fit. The most commonly used software in spatial restraint-based modeling is MODELLER
MODELLER
MODELLER is a computer program used in producing homology models of protein tertiary structures as well as quaternary structures . It implements a technique inspired by nuclear magnetic resonance known as satisfaction of spatial restraints, by which a set of geometrical criteria are used to create...

 and a database called ModBase has been established for reliable models generated with it.

Loop modeling


Regions of the target sequence that are not aligned to a template are modeled by loop modeling
Loop modeling
Loop modeling is a problem in protein structure prediction requiring the prediction of the conformations of loop regions in proteins without the use of a structural template. The problem arises often in homology modeling, where the tertiary structure of an amino acid sequence is predicted based on...

; they are the most susceptible to major modeling errors and occur with higher frequency when the target and template have low sequence identity. The coordinates of unmatched sections determined by loop modeling programs are generally much less accurate than those obtained from simply copying the coordinates of a known structure, particularly if the loop is longer than 10 residues. The first two sidechain dihedral angle
Dihedral angle
In geometry, a dihedral or torsion angle is the angle between two planes.The dihedral angle of two planes can be seen by looking at the planes "edge on", i.e., along their line of intersection...

s (χ1 and χ2) can usually be estimated within 30° for an accurate backbone structure; however, the later dihedral angles found in longer side chains such as lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

 and arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...

 are notoriously difficult to predict. Moreover, small errors in χ1 (and, to a lesser extent, in χ2) can cause relatively large errors in the positions of the atoms at the terminus of side chain; such atoms often have a functional importance, particularly when located near the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

.

Model assessment


Assessment of homology models without reference to the true target structure is usually performed with two methods: statistical potential
Statistical potential
In protein structure prediction, a statistical potential or knowledge-based potential is an energy function derived from an analysis of known protein structures in the Protein Data Bank....

s or physics-based energy calculations. Both methods produce an estimate of the energy (or an energy-like analog) for the model or models being assessed; independent criteria are needed to determine acceptable cutoffs. Neither of the two methods correlates exceptionally well with true structural accuracy, especially on protein types underrepresented in the PDB
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....

, such as membrane protein
Membrane protein
A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes.-Function:...

s.

Statistical potentials are empirical methods based on observed residue-residue contact frequencies among proteins of known structure in the PDB. They assign a probability or energy score to each possible pairwise interaction between amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

s and combine these pairwise interaction scores into a single score for the entire model. Some such methods can also produce a residue-by-residue assessment that identifies poorly scoring regions within the model, though the model may have a reasonable score overall. These methods emphasize the hydrophobic core and solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

-exposed polar
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...

 amino acids often present in globular protein
Globular protein
Globular proteins, or spheroproteins are one of the two main protein classes, comprising "globe"-like proteins that are more or less soluble in aqueous solutions...

s. Examples of popular statistical potentials include Prosa
Prosa
Prosa is Norway's largest literary magazine. It is a magazine dealing with prose, academic literature, writing culture, and cultural politics, and contains literary essays, reviews, and articles. The magazine prides itself on its editorial independence, and is published by the Norwegian Non-Fiction...

 and DOPE
DOPE score
DOPE, or Discrete Optimized Protein Energy, is a statistical potential used to assess homology models in protein structure prediction...

. Statistical potentials are more computationally efficient than energy calculations.

Physics-based energy calculations aim to capture the interatomic interactions that are physically responsible for protein stability in solution, especially van der Waals
Van der Waals force
In physical chemistry, the van der Waals force , named after Dutch scientist Johannes Diderik van der Waals, is the sum of the attractive or repulsive forces between molecules other than those due to covalent bonds or to the electrostatic interaction of ions with one another or with neutral...

 and electrostatic interactions. These calculations are performed using a molecular mechanics
Molecular mechanics
Molecular mechanics uses Newtonian mechanics to model molecular systems. The potential energy of all systems in molecular mechanics is calculated using force fields...

 force field
Force field (chemistry)
In the context of molecular modeling, a force field refers to the form and parameters of mathematical functions used to describe the potential energy of a system of particles . Force field functions and parameter sets are derived from both experimental work and high-level quantum mechanical...

; proteins are normally too large even for semi-empirical quantum mechanics
Quantum mechanics
Quantum mechanics, also known as quantum physics or quantum theory, is a branch of physics providing a mathematical description of much of the dual particle-like and wave-like behavior and interactions of energy and matter. It departs from classical mechanics primarily at the atomic and subatomic...

-based calculations. The use of these methods is based on the energy landscape
Energy landscape
In physics, an energy landscape is a mapping of all possible conformations of a molecular entity, or the spatial positions of interacting molecules in a system, and their corresponding energy levels, typically Gibbs free energy, on a two- or three-dimensional Cartesian coordinate system.In...

 hypothesis of protein folding, which predicts that a protein's native state
Native state
In biochemistry, the native state of a protein is its operative or functional form. While all protein molecules begin as simple unbranched chains of amino acids, once completed they assume highly specific three-dimensional shapes; that ultimate shape, known as tertiary structure, is the folded...

 is also its energy minimum. Such methods usually employ implicit solvation
Implicit solvation
Implicit solvation is a method of representing solvent as a continuous medium instead of individual “explicit” solvent molecules most often used in molecular dynamics simulations and in other applications of molecular mechanics...

, which provides a continuous approximation of a solvent bath for a single protein molecule without necessitating the explicit representation of individual solvent molecules. A force field specifically constructed for model assessment is known as the Effective Force Field (EFF) and is based on atomic parameters from CHARMM
CHARMM
CHARMM is the name of a widely used set of force fields for molecular dynamics as well as the name for the molecular dynamics simulation and analysis package associated with them...

.

A very extensive model validation report can be obtained using the Radboud Universiteit Nijmegen "What Check" software which is one option of the Radboud Universiteit Nijmegen "What If" software package; it produces a many page document with extensive analyses of nearly 200 scientific and administrative aspects of the model. "What Check" is available as a free server; it can also be used to validate experimentally determined structures of macromolecules.

One newer method for model assessment relies on machine learning
Machine learning
Machine learning, a branch of artificial intelligence, is a scientific discipline concerned with the design and development of algorithms that allow computers to evolve behaviors based on empirical data, such as from sensor data or databases...

 techniques such as neural nets, which may be trained to assess the structure directly or to form a consensus among multiple statistical and energy-based methods. Very recent results using support vector machine
Support vector machine
A support vector machine is a concept in statistics and computer science for a set of related supervised learning methods that analyze data and recognize patterns, used for classification and regression analysis...

 regression on a jury of more traditional assessment methods outperformed common statistical, energy-based, and machine learning methods.

Structural comparison methods


The assessment of homology models' accuracy is straightforward when the experimental structure is known. The most common method of comparing two protein structures uses the root-mean-square deviation (RMSD) metric to measure the mean distance between the corresponding atoms in the two structures after they have been superimposed. However, RMSD does underestimate the accuracy of models in which the core is essentially correctly modeled, but some flexible loop regions are inaccurate. A method introduced for the modeling assessment experiment CASP
CASP
CASP, which stands for Critical Assessment of Techniques for Protein Structure Prediction, is a community-wide, worldwide experiment for protein structure prediction taking place every two years since 1994...

 is known as the global distance test
Global distance test
The global distance test or GDT is a measure of similarity between two protein structures with identical amino acid sequences but different tertiary structures...

 (GDT) and measures the total number of atoms whose distance from the model to the experimental structure lies under a certain distance cutoff. Both methods can be used for any subset of atoms in the structure, but are often applied to only the alpha carbon
Alpha carbon
The alpha carbon in organic chemistry refers to the first carbon that attaches to a functional group . By extension, the second carbon is the beta carbon, and so on....

 or protein backbone atoms to minimize the noise created by poorly modeled side chain rotameric states, which most modeling methods are not optimized to predict.

Benchmarking


Several large-scale benchmarking
Benchmarking
Benchmarking is the process of comparing one's business processes and performance metrics to industry bests and/or best practices from other industries. Dimensions typically measured are quality, time and cost...

 efforts have been made to assess the relative quality of various current homology modeling methods. CASP
CASP
CASP, which stands for Critical Assessment of Techniques for Protein Structure Prediction, is a community-wide, worldwide experiment for protein structure prediction taking place every two years since 1994...

 is a community-wide prediction experiment that runs every two years during the summer months and challenges prediction teams to submit structural models for a number of sequences whose structures have recently been solved experimentally but have not yet been published. Its partner CAFASP
CAFASP
CAFASP, or the Critical Assessment of Fully Automated Structure Prediction, is a large-scale blind experiment in protein structure prediction that studies the performance of automated structure prediction webservers in homology modeling, fold recognition, and ab initio prediction of protein...

 has run in parallel with CASP but evaluates only models produced via fully automated servers. Continuously running experiments that do not have prediction 'seasons' focus mainly on benchmarking publicly available webservers. LiveBench
LiveBench
LiveBench is a continuously running benchmark project for assessing the quality of protein structure prediction and secondary structure prediction methods. LiveBench focuses mainly on homology modeling and protein threading but also includes secondary structure prediction, comparing publicly...

 and EVA
EVA
Eva or EVA may refer to:* Eva , a given name for women** Eva , a list of people with the name EvaIt may also refer to:-In business and economics:* Earned Value Analysis, a measurement of project progress...

 run continuously to assess participating servers' performance in prediction of imminently released structures from the PDB. CASP and CAFASP serve mainly as evaluations of the state of the art in modeling, while the continuous assessments seek to evaluate the model quality that would be obtained by a non-expert user employing publicly available tools.

Accuracy


The accuracy of the structures generated by homology modeling is highly dependent on the sequence identity between target and template. Above 50% sequence identity, models tend to be reliable, with only minor errors in side chain
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...

 packing and rotameric state, and an overall RMSD between the modeled and the experimental structure falling around 1 Â
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

. This error is comparable to the typical resolution of a structure solved by NMR. In the 30–50% identity range, errors can be more severe and are often located in loops. Below 30% identity, serious errors occur, sometimes resulting in the basic fold being mis-predicted. This low-identity region is often referred to as the "twilight zone" within which homology modeling is extremely difficult, and to which it is possibly less suited than fold recognition methods.

At high sequence identities, the primary source of error in homology modeling derives from the choice of the template or templates on which the model is based, while lower identities exhibit serious errors in sequence alignment that inhibit the production of high-quality models. It has been suggested that the major impediment to quality model production is inadequacies in sequence alignment, since "optimal" structural alignment
Structural alignment
Structural alignment attempts to establish homology between two or more polymer structures based on their shape and three-dimensional conformation. This process is usually applied to protein tertiary structures but can also be used for large RNA molecules...

s between two proteins of known structure can be used as input to current modeling methods to produce quite accurate reproductions of the original experimental structure.

Attempts have been made to improve the accuracy of homology models built with existing methods by subjecting them to molecular dynamics
Molecular dynamics
Molecular dynamics is a computer simulation of physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a period of time, giving a view of the motion of the atoms...

 simulation in an effort to improve their RMSD to the experimental structure. However, current force field
Force field (chemistry)
In the context of molecular modeling, a force field refers to the form and parameters of mathematical functions used to describe the potential energy of a system of particles . Force field functions and parameter sets are derived from both experimental work and high-level quantum mechanical...

 parameterizations may not be sufficiently accurate for this task, since homology models used as starting structures for molecular dynamics tend to produce slightly worse structures. Slight improvements have been observed in cases where significant restraints were used during the simulation.

Sources of error


The two most common and large-scale sources of error in homology modeling are poor template selection and inaccuracies in target-template sequence alignment. Controlling for these two factors by using a structural alignment
Structural alignment
Structural alignment attempts to establish homology between two or more polymer structures based on their shape and three-dimensional conformation. This process is usually applied to protein tertiary structures but can also be used for large RNA molecules...

, or a sequence alignment produced on the basis of comparing two solved structures, dramatically reduces the errors in final models; these "gold standard" alignments can be used as input to current modeling methods to produce quite accurate reproductions of the original experimental structure. Results from the most recent CASP experiment suggest that "consensus" methods collecting the results of multiple fold recognition and multiple alignment searches increase the likelihood of identifying the correct template; similarly, the use of multiple templates in the model-building step may be worse than the use of the single correct template but better than the use of a single suboptimal one. Alignment errors may be minimized by the use of a multiple alignment even if only one template is used, and by the iterative refinement of local regions of low similarity.
A lesser source of model errors are errors in the template structure. The PDBREPORT database lists several million, mostly very small but occasionally dramatic, errors in experimental (template) structures that have been deposited in the PDB
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....

.

Serious local errors can arise in homology models where an insertion or deletion mutation or a gap in a solved structure result in a region of target sequence for which there is no corresponding template. This problem can be minimized by the use of multiple templates, but the method is complicated by the templates' differing local structures around the gap and by the likelihood that a missing region in one experimental structure is also missing in other structures of the same protein family. Missing regions are most common in loops where high local flexibility increases the difficulty of resolving the region by structure-determination methods. Although some guidance is provided even with a single template by the positioning of the ends of the missing region, the longer the gap, the more difficult it is to model. Loops of up to about 9 residues can be modeled with moderate accuracy in some cases if the local alignment is correct. Larger regions are often modeled individually using ab initio structure prediction techniques, although this approach has met with only isolated success.

The rotameric states of side chains and their internal packing arrangement also present difficulties in homology modeling, even in targets for which the backbone structure is relatively easy to predict. This is partly due to the fact that many side chains in crystal structures are not in their "optimal" rotameric state as a result of energetic factors in the hydrophobic core and in the packing of the individual molecules in a protein crystal. One method of addressing this problem requires searching a rotameric library to identify locally low-energy combinations of packing states. It has been suggested that a major reason that homology modeling so difficult when target-template sequence identity lies below 30% is that such proteins have broadly similar folds but widely divergent side chain packing arrangements.

Utility


Uses of the structural models include protein–protein interaction prediction, protein–protein docking, molecular docking, and functional annotation of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

s identified in an organism's genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....

. Even low-accuracy homology models can be useful for these purposes, because their inaccuracies tend to be located in the loops on the protein surface, which are normally more variable even between closely related proteins. The functional regions of the protein, especially its active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

, tend to be more highly conserved and thus more accurately modeled.

Homology models can also be used to identify subtle differences between related proteins that have not all been solved structurally. For example, the method was used to identify cation binding site
Binding site
In biochemistry, a binding site is a region on a protein, DNA, or RNA to which specific other molecules and ions—in this context collectively called ligands—form a chemical bond...

s on the Na+/K+ ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...

 and to propose hypotheses about different ATPases' binding affinity. Used in conjunction with molecular dynamics
Molecular dynamics
Molecular dynamics is a computer simulation of physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a period of time, giving a view of the motion of the atoms...

 simulations, homology models can also generate hypotheses about the kinetics and dynamics of a protein, as in studies of the ion selectivity of a potassium
Potassium
Potassium is the chemical element with the symbol K and atomic number 19. Elemental potassium is a soft silvery-white alkali metal that oxidizes rapidly in air and is very reactive with water, generating sufficient heat to ignite the hydrogen emitted in the reaction.Potassium and sodium are...

 channel. Large-scale automated modeling of all identified protein-coding regions in a genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....

 has been attempted for the yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...

 Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

, resulting in nearly 1000 quality models for proteins whose structures had not yet been determined at the time of the study, and identifying novel relationships between 236 yeast proteins and other previously solved structures.

See also

  • Protein structure prediction
    Protein structure prediction
    Protein structure prediction is the prediction of the three-dimensional structure of a protein from its amino acid sequence — that is, the prediction of its secondary, tertiary, and quaternary structure from its primary structure. Structure prediction is fundamentally different from the inverse...

  • Protein structure prediction software
  • Protein threading