RING - Residue Interaction Network Generator

Network definitions

Network based on C-alpha carbon distances

Link definitions

This type of network defineds a link between a pair of non-adjacent residues if the distance between their alpha carbons is ≤ the threshold value. This threshold can be changed by introducing a new value in the form cutoff. The definition implies that only one connection between a pair of residues can be traced (dashed red line in the image below).
NB: The new value can be integer or decimal, and "." should be used as decimal separator.

Network attributes

This type of network can generate 11 different types of edge attributes and 12 node attributes. Once loaded into CYTOSCAPE, these attributes will be visible in the data panel when you select one or more connections or nodes. The edge attributes are:

• Atoms that determine the interaction (C-alpha in this case).
• Distance between these atoms.
• If there is at least one hydrogen bond between the pair of residues defined in contact.
• If the pair in contact form a salt bridge.
• If the pair in contact is involved in a π-cation interaction.
• If there is a π-π interaction between the pair in contact.
• If the pair in contact form a disulfide bond.
• The site of interaction.
• The weight of the connection (see below).
• The mutual information between residue pairs (see below).
• The APC mutual information correction between residue pairs (see below).

The node attributes are:

• Type of secondary structure in which the residue is involved. Defined from the φ and ψ torsion angles (three classes) and by DSSP in three and eight classes.
• Solvent Accessibility as computed by DSSP.
• Conservation indicates which is the conservation of each amino acid in the sequence in the PSI-BLAST generated multiple sequence alignment.
• FRST Energy indicates which is the energy determined by FRST of each amino acid in the sequence.
• TAP Score Energy indicates which is the energy determined by TAP of each amino acid in the sequence.
• Complete node ID (see file format section).
• Node degree in the network.
• C&alpha B-factor of the residue.
• C&alpha Occupancy of the residue.
• Cumulative mutual information of the residue, as the sum of the mutual information with all the other amino acids with which it is connected (see below).

For explanations about the meaning of the attributes listed see the explanation paragraphs for the respective methods.

A network of this type can be useful for coarse inspection; attributes only help you to remember what types of strong interactions exist between the residues in contact, without wanting to specify anything else.

Network based on distances between the closest atoms

Link definitions

In this type of network a link is defined if the distance (in Å) between a pair of non-adjacent residues and their closest atoms is ≤ than the threshold value. The threshold can be changed by introducing a new value in the form cutoff, (dashed red line in the image).

If the closest atoms determining a link between a pair of residues form a hydrogen bond, then the connection is classified as a hydrogen bond (dashed red line in the image across). However, unlike the previous case, if the same pair of residues are found involved in other hydrogen bonds that do not involve the closest atom, more directed links are added (dashed blue line in the image across). This means that the couples involved in several hydrogen bonds will be represented by pairs of nodes with multiple links.

Network attributes

For this type of network there are 29 different types of edge attributes stored in as many files and 12 node attributes. Once loaded into CYTOSCAPE, these attributes will be visible in the data panel when selecting one or more connections. The edge attributes are:

• Atoms that determine the interaction (the closest atoms in this case).
• Distance between these atoms.
• If there is at least one hydrogen bond between the pair of residues defined in contact.
• 3-letters code of the hydrogen bond donor amino acid – 3-letters code of the hydrogen bond acceptor amino acid.
• Atomic code of the donor atom – atomic code of the acceptor atom.
• Distance between donor and acceptor atoms.
• Angle between the donor and acceptor atoms, with vertex on the hydrogen atom.
• If the pair in contact form a salt bridge.
• 3-letters code of the positively charged amino acid – 3-letters code of the negatively charged amino acid.
• Distance between the mass centers of charges.
• Angle of the bridge ρ.
• If there is a π-π interaction between the pair in contact.
• The value of n.
• The value of p.
• The value of θ.
• The spatial configuration of the pair.
• If the pair in contact is involved in a π-cation interaction.
• 3-letters code of the positively charged amino acid – 3-letters code of the aromatic amino acid.
• Distance between the mass centers of the cation and the closest atom of the aromatic system.
• Angle α between the cation and the π system.
• If it is present a guanidine ion.
• Mutual disposition of the aromatic system and guanidine ion.
• If the pair in contact form a disulfide bond.
• Calculated distance of the bridge.
• Angle χ.
• Weight of the connection (see below).
• Site of interaction.
• The mutual information between residue pairs (see below).
• The APC mutual information correction between residue pairs (see below).

The node attributes are:

• Type of secondary structure in which the residue is involved. Defined from the φ and ψ torsion angles (three classes) or by DSSP in three or eight classes.
• Solvent Accessibility as computed by DSSP.
• Conservation indicates which is the conservation of each amino acid in the sequence in the PSI-BLAST generated multiple sequence alignment.
• FRST Energy indicates which is the energy determined by FRST of each amino acid in the sequence.
• TAP Score Energy indicates which is the energy determined by TAP of each amino acid in the sequence.
• Complete node ID (see file format section).
• Node degree in the network.
• C&alpha B-factor of the residue.
• C&alpha Occupancy of the residue.
• Cumulative mutual information of the residue, as the sum of the mutual information with all the other amino acids with which it is connected (see below).

For explanations about the meaning of the attributes listed see the explanation paragraphs for the respective methods. This type of network useful when one wants to analyze interactions that are present within a protein on the base of the distances between residues.

Network based on van der Waals contacts

Link definitions

To generate this type of network RING uses PROBE to identify the van der Waals interactions between the atoms of each pair of considered residues and defines the two residues as in contact if between any of their atoms exists at least one van der Waals interaction.

In this case, as many links as the van der Waals interactions that are identified between the same pair of residues defined in contact are added. RING subsequently identifies all the other types of noncovalent interactions in the constructed network, and adds the necessary connections for them.
As above, if the same pair of residues are found involved in several hydrogen bonds, the same number links are added to the nodes that identify them.

Network attributes

For this type of network there are 30 different types of edge attributes stored in as many files and 12 node attributes. Once loaded into CYTOSCAPE, these attributes will be visible in the data panel when selecting one or more connections. The edge attributes are:

• The closest atoms.
• Distance between these atoms.
• If there is at least one hydrogen bond between the pair of residues defined in contact.
• 3-letters code of the hydrogen bond donor amino acid – 3-letters code of the hydrogen bond acceptor amino acid.
• Atomic code of the donor atom – atomic code of the acceptor atom.
• Distance between donor and acceptor atoms.
• Angle between the donor and acceptor atoms, with vertex on the hydrogen atom.
• If the pair in contact form a salt bridge.
• 3-letters code of the positively charged amino acid – 3-letters code of the negatively charged amino acid.
• Distance between the mass centers of charges.
• Angle of the bridge ρ.
• If there is a π-π interaction between the pair in contact.
• The value of n.
• The value of p.
• The value of θ.
• The spatial configuration of the pair.
• If the pair in contact is involved in a π-cation interaction.
• 3-letters code of the positively charged amino acid – 3-letters code of the aromatic amino acid.
• Distance between the mass centers of the cation and the closest atom of the aromatic system.
• Angle α between the cation and the π system.
• If it is present a guanidine ion.
• Mutual disposition of the aromatic system and guanidine ion.
• If the pair in contact form a disulfide bond.
• Calculated distance of the bridge.
• Angle χ.
• Weight of the connection (see below).
• Van der Waals contact score (see below).
• Site of interaction.
• The mutual information between residue pairs (see below).
• The APC mutual information correction between residue pairs (see below).

The node attributes are:

• Type of secondary structure in which the residue is involved. Defined from the φ and ψ torsion angles (three classes) or by DSSP in three or eight classes.
• Solvent Accessibility as computed by DSSP.
• Conservation indicates which is the conservation of each amino acid in the sequence in the PSI-BLAST generated multiple sequence alignment.
• FRST Energy indicates which is the energy determined by FRST of each amino acid in the sequence.
• TAP Score Energy indicates which is the energy determined by TAP of each amino acid in the sequence.
• Complete node ID (see file format section).
• Node degree in the network.
• C&alpha B-factor of the residue.
• C&alpha Occupancy of the residue.
• Cumulative mutual information of the residue, as the sum of the mutual information with all the other amino acids with which it is connected (see below).

For explanations about the meaning of the attributes listed see the explanation paragraphs for the respective methods. This type of network is very useful when one wants to analyze the actual number of interactions present within a protein.

Connection weights

RING assigns to each established connection a weight, with the exception od peptide bonds when these are generated. This weight can be used to calculate some topological parameters for the generated network (like shortest path betweenness centrality, closeness centrality, shortest path degree centrality...) using some CYTOSCAPE plug-ins available on the web.

Weights for the closest atom networks

The attribute weight generated by RING for the closest atoms network correspond to the approximate average free energy of each interaction and represent the average values used in the literature arising from both simulations and experimental data. These values are:

For the disulfide bridge free energy the value used is the dissociation enthalpy of this covalent bond. For simple interactions (i.e. between closest atoms among which there is no other classified interactions) we consider the average attractiveness component of the van der Waals force that exist between any two residues.

Weights for the van der Waals networks

The attribute weight generated by RING for the van der Waals networks is the same generated for the closatom network (see above). In addition the edge attribute van der Waals contact score is used as weight of the van der Waals interactions. The van der Waals contact score is generated for every van der Waals interaction as follows:
Van der Waals interactions identified for the same pair of residues are classified according to the interaction site which includes the interacting atoms: mc-mc, mc-sc, sc-sc.
for each set of interactions is done the sum of the scores provided by PROBE for each of them: &sum(mc-mc), &sum(mc-sc), &sum(sc-sc).
total score from each sum is given to all the van der Waals interactions that belong to the same site of interaction.

For the C-alpha network the attribute weight generated by RING is = 1 for all interactions. This is because in such type of network, atoms that determine the interactions (C-alpha) can be affected at most by the van der Waals force which is very small because generally, there is a large distance between pairs of them.

HBexplore criteria

HBexplore provides 2 geometric criteria to identify potential hydrogen bonds.

First criterion

The first and simplest is based on constraints with respect to distances between the acceptor atom A and the hydrogen atom H, called d₁, and between donor atom D and acceptor atom A called d₂. The following are calculated: the angles between the bond -D-H and d₁, called α; the link between A-A₁- (where A₁ is the atom which is covalently linked to the acceptor) and d₁, called γ; the angle between A-A1 and d₂, called β. If the acceptor atom A is part of a ring, then it will have two neighbors, A₁ and A₂. In this case the atom which is covalently linked to A (i.e., A_m) is lying in the middle point along the line joining A₁ and A₂, (see the image across). For donor atoms with sp³ hybridization the distance H-A is first determined but only after the other geometrical parameters are calculated. In RING the threshold used is the same as in HBexplore: d₁ < 2.5 Å, d₂ < 3.9 Å, α > 90°, β > 90° γ > 90°.

Second criterion

The second criterion is based on the directionality of the hybrid orbitals and it arises from the observation that many hydrogen bonds show a clear directionality. This comes from the overlap of hybrid orbitals because while the s orbital of the donor has a spherical symmetry, the hybrid orbital of the acceptor atom in sp₂ or sp₃ configuration has an angle of 120° and 109° respectively. In HBexplore, the orbitals are treated as vectors whose direction is inferred from the atoms surrounding the acceptor. To constrain the angle DHA, the angle between H-A and the direction of acceptor hybrid orbitals (Hyb) for which there must be at least one with an angle less than 60° are also taken into account (see the image across). This criterion considers as donor atoms only O, N and sulfur S while the C is not covered. RING uses the standard threshold used by the HBexplore: d₁ < 2.5 Å, d₂ < 3.9 Å, α > 90°, β > 90°, ε < 60°.

RING saves each hydrogen bond characteristics in order to have a directed edge in the graph that will be generated. The connection will originate from the node that represents the amino acid with donor atom and ends on the node that represents the amino acid with the acceptor atom.

Method to find salt bridges

RING investigates on the presence or absence of a salt bridge when a residue is usually negatively charged at physiological pH. Asp or Glu, is usually set in salt contact with a usually positively charged residues like Arg, Lys or His.
Two residues with opposite charges are considered involved in a salt bridge if the distance between the mass centers of the charged groups in their side chains are ≤ the distance threshold in Å.
If the user does not set any threshold, a threshold = 4 Å is used by default. This value was empirically derived from the analysis of a large set of protein structures

RING calculates the center of mass of the charged group for the following residues:

• For Lys the mass center of the positive charge is identified with the coordinates of the N_ξ atom.
  

• For Asp and Glu the negative charge is considered delocalized between the three atoms forming the ionized side chain carboxylic group. Thus the center of mass is placed between C and the two O atoms.
  
  
• For the Arg the positive charge is considered delocalized on the three N of the guanidine group in the side chain. Thus the mass center of the charge is identified with the coordinates of the C_ξ in the guanidine group.
  

• Finally His has two N-H titratable groups in its side chain imidazole ring (N_δ1 and N_δ2). Although the N_δ2 is thought to be especially protonated (i.e. the farthest from the main chain), the charge is considered as delocalized, lying in the center of mass between atoms N_δ1, C_ε1, N_δ2.
  

RING also calculates the angle ε of the bridge, in degrees, defined as the angle formed between the two lines connecting the C-alpha with the mass center of the charged group, respectively in each pair of residues (see ρ in image above).
RING saves the characteristics of each salt bridge in order to have a directed edge on the generated graph. In other words the connection originates in the node that represents the positively charged amino acid and ends on the node representing the negatively charged amino acid.

Method to find π - π interactions

Given a pair of residues defined as being in contact, RING checks if they both belong to the category of aromatic amino acids (His, Tyr, Trp, Phe). Then RING states if a π-π interaction exists between them if there is at least one pair of atoms belonging to one of the two side chains at distance < 6 Å apart (see image across). This default threshold value can be changed by entering a new value in the field π-π minimum distance. RING subsequently tries to determine the reciprocal position of the two aromatic rings considered using a combination of spatial and rotational orientation.

Given a pair of aromatic systems i and j, RING establishes a cartesian coordinate system originating from the mass center of the i ring. The axis passing through Cγ and Cγ is defined as X axis, so the plane of the ring becomes the XY plane, while the Z axis originates from the mass center and lies perpendicular to this plane. RING defines three spatial orientations: above, above-aside, aside on the basis of the two distances denoted n and p:

• n represents the distance in Å between the origin of the orthogonal system (the ring centroid) and the projection of the mass center of the coupled ring j on the Z-axis of the defined orthogonal system;

• p is the distance in Å between the origin of the orthogonal system and the projection of the mass center of the ring j on the XY plane.

RING also defines three rotational orientations:

• Parallel (P).

• Tilted (T).

• Perpendicular (N).

These rotational orientations are defined on the basis of the dihedral angle θ (the angle in degrees between the planes of the two rings). The rotational and spatial orientations are combined to define four spatial configurations of the two rings:

• Parallel orientation if 0° ≤θ < 30° or 150° < θ ≤180°.

• T-orientation with the edge to face if p < 3.5 Å and 30° ≤θ ≤ 150°.

• T-orientation with the face to the edge if p ≥ 3.5 Å, n < 3 Å and 30° ≤θ ≤150°.

• L-orientation (i.e. where the two rings are arranged resembling the letter "L") if p ≥ 3.5 Å, n ≥ 3 Å and 30° ≤θ ≤ 150°.

The constraint on the minimum distance between side chains that identifies a π-π interaction was derived empirically from the analysis of a large group of protein structures. It has to be noticed that there are more accurate methods to determine this interaction but these are computationally more expensive and only small improvements are reported.

As for the remaining constraints on n, p and θ the solution here is widely adopted in the literature to evaluate the reciprocal position of aromatic systems.

To calculate π-system mass centers, the following rules are adopted:

• For Tyr and Phe the 6 atoms of the benzene ring are considered in the canter of mass calculation. The hydroxyl oxygen of Tyr is not considered, because it has shown that it does not change the interaction propensity for this residue.

• For His RING considers the 5 atoms of the imidazole ring.

• For Trp the 9 atoms of the indole ring are considered.

For this type of interaction the links are not directed.

Method to find π - cation interactions

RING searches for π-cation interactions when two residues in contact are respectively an amino acid positively charged at physiological pH (Arg or Lys) and an amino acid with aromatic side chain (Phe, Tyr, Trp). His, although possessing an aromatic side chain, is not considered because it can participate in this type of interaction both as cation and as a π-system, depending on its protonation state.

To identify a π-cation interaction two conditions must be met:

• The mass center of the charged group in the side chain of a cation should be located at a distance ≤ the threshold distance specified (π-cation minimum distance) to any atom of the other residue in the π-system. If the user does not enter a value a threshold of 7 Å is used as default.

• α is defined as the angle in degrees between the cation mass center and the vector normal to the aromatic ring plane passing through its mass center, α must be: 0° ≤ α ≤60° and 120° ≤ α ≤180°.

The default thresholds for the distance and angle are empirically derived. These constraints intend to select those cations whose positive charge lies above or below the ring area, where the ring surface potential is negative. This criterion is approximate but sufficient for our purposes and not very expensive in terms of computational time.

Moreover, given the different intensity of interaction between Arg and a π-system according to the orientation of the guanidinium ion, RING divides this interaction into three categories. The catagories depend on the dihedral angle θ in degrees defined between the plane of the aromatic ring and the cationic guanidine group:

• Planar when 0° ≤ θ < 30° or 150° < θ ≤180°.

• Oblique when 30° ≤ θ ≤60° or 120° ≤ θ ≤150°.

• Orthogonal when 60° < θ < 120°.

The rules to calculate the mass center of charged group in Lys and Arg are the same as in the method used to search for salt bridges (see above), i.e. for N_ζ Lys and C_ζ of the guanidine group in Arg. For the calculation of mass centers for the aromatic systems the same rules are applied as defined for π-π interactions (except in the case of His because it is not considered (see above)).

RING saves the characteristics of each π-cation interaction in order to to have directed edges in the graph. Edges originate from the positively charged node and end on the the aromatic node.

Method to find disulfide bridge

Being a covalent bond between two atoms, disulfide bridges present a constant distance. RING uses this type of constraint to identify disulfide bridges between two Cys residues being in contact.

Two Cys are considered covalently bounded if the distance between their sulfur atoms (S_γ) is ≤ the distance threshold (d in image across). If the user does not place any value, 3 Å is used as default threshold. This threshold was derived empirically.

Furthermore RING calculates the virtual angle in degrees between the vectors joining the C_β and S_γ of the two Cys residues called χ, which can provide information on the bridge geometry.

For this type of interaction corresponding links in the graph are not directed.

Addition of hydrogen atoms by REDUCE

The correct orientation of Asn and Gln side chain -NH₂ against O atoms is very important when these residues are involved in hydrogen bonds. This is especially true when these hydrogen bonds occur in the active site of a protein or when examining the network of hydrogen bonds in a protein.

To add the hydrogen atoms coordinates to molecular structures files and to correct the orientation errors of important chemical groups in the side chain of certain amino acids, RING relies on the external program REDUCE.

REDUCE first assigns the correct orientation of amide groups in side chains of Asn and Gln residues. It then optimizes the orientation of the hydroxyl groups (-OH), sulphydrate (-SH), amino (-NH₃⁺), methyl in Met and correctly orients the imidazole ring in His. REDUCE's next step is to add the coordinates of the missing hydrogen atoms consistently with the new conformations assigned to the side chains.

REDUCE implements a simple algorithm which takes into account collisions between the van der Waals volumes of the atoms belonging to the amino acid side chain groups involved. It analyzes possible local networks of potential hydrogen bonds.

Briefly the procedure adopted by REDUCE is:

1. First it identifies and fixes the orientation of all the ligands or metal groups covalently bounded in the structure.

2. Then all group pairs that can potentially interact considering the entire range of positions of hydrogen atoms in both orientations of the side chains rotable groups are identified.

3. For each position, all possible clashes between the van der Waals volumes which would be verified are evaluated.

4. The best conformation for the involved group is chosen (the conformation which preserves interaction and minimizes collisions).

5. The chosen conformation is optimized and fixed in a new PDB file with the coordinates of hydrogen atoms calculated at the end of the process. This PDB file is made available to the user on the results page.

Calculation of van der Waals interactions by PROBE.

The program PROBE allows one to evaluate atomic packing within molecules. PROBE reads atomic coordinates from protein databank (PDB) format files and generate contact dot-lists where atoms are in close contact. Alternatively, the packing information can be quantified and displayed in a table listing scores for van der Waals interactions, H-bonds and atomic overlaps ("clashes"). Essentially, the approach is to place a very small probe (typically of radius 0.25Å) at points along the van der Waals surface of a selected set of atoms and determine if this probe also contacts atoms within a second "target" set. So PROBE provides a flexible method for selecting the source and target atoms.

Analysis of molecular contact surfaces by PROBE requires that ALL atoms are considered for this, before using PROBE, the PDB file must be submitted to the program REDUCE to add hydrogens to the coordinate file (see above).

The non-overlapping van der Waals contacts are quantified by an error-function weighting, which awards close contacts a higher score than distant or significantly overlapping ones, but slight overlaps are still favorable in net effect. The combined score is a weighted sum of the weighted non-overlapping van der Waals contacts, the volume of hydrogen bonds and the volume of overlaps. The PROBE program summarizes these scoring data for all parts of an entire structure and can output a file with information for every atom or residue.

RING runs PROBE on the PDB file containing coordinates of all hydrogen atoms added by REDUCE and uses the contact list generated by it to get information about the van der Waals interactions between pairs of residues defined in contact. RING also catch the scores of each interaction to formulate a weighted score for each of them, depending on the site where interaction occurs (see above).

Method for calculating the filtered sequence based on the nodes degree

The degree of a node is its total number of edges (i.e. the number of neighbors). When generated, peptidic bonds are not used to compute node degrees. Taking into account the important role that could be played in a protein by amino acids with high connectivity in the network (the so-called hubs), RING produces a file containing a multiple sequence alignment in FASTA format, built on the amino acid sequence of the protein being examined, in which each sequence is filtered according to the increasing degree of the corresponding nodes.
This method does not take into account the distinction between grade-in and grade-out as is considered in directed graph theory.

Once the number of interactions for each node (their deg(n)) is calculated, RING generates several sequences filtered based on this value. Sequences filtered using a threshold level deg_t produces the letter corresponding to the corresponding amino acid a only if g(a) ≤ deg_t, otherwise the symbol x is displayed in the correspondent position.

In some cases, it may happen that the SEQRES sequence corresponding to the residues actually present in the strucure, lacks some amino acids. In this case, RING tries to determine the full length of the original protein sequence based on the present residues numbering and fills any sequence gaps with the symbol -.

The check box Node degree sequence alignment enables the calculation of this multiple sequence alignment by RING. If Node degree sequence alignment is selected, the option Starting threshold allows the user to introduce the threshold degree from witch to start the construction of the multialignment. All sequences are filtered with a default value starting from degree = 1.

The multiple sequence alignment can be easily loaded and manipulated in JALVIEW, which is a useful tool to visually capture which amino acids act as hubs according to the network (see output page). These residues are those which persist longer in the multiple sequence alignment (i.e. they are more evolutionary conserved). Along with the alignment RING also produces a text file with extension .anal containing useful information about nodes degree. For details about the reported information, go to the output page. This file can be useful for the network connectivity analysis.

Secondary Structure and Solvent Accessibility

DSSP (The Dictionary of Protein Secondary Structure) is a commonly used program to describe proteins secondary structure. Among many other features it also provides solvent accesibilty (Ų). DSSP assigns secondary structure based on hydrogen bonding patterns. Hydrogen bonds are defined by DSSP using an electrostatic model. DSSP assigns a charge of ± q₁ = 0.42e to the carbonyl carbon hydrogen (+) and oxygen respectively (-), and charges of ± q₂ =0.20e to the amide nitrogen (-) and hydrogen (+), respectively. The electrostatic energy (E) is:

E = q₁ q₂ f ( ¹/_rON + ¹/_rHC' - ¹/_rOH - ¹/_rNC'} ).

where f = 332 kcal/mol and r_XX are the respecitve distances. Hydrogen bonds only exists if E is less than -0.5 kcal/mol for a given pair donnor acceptor. The eigth secondary structure classes are:

• G = 3-turn helix (3-10 helix). Minimum length 3 residues.
• H = 4-turn helix (alpha helix). Minimum length 4 residues.
• I = 5-turn helix (π helix). Min length 5 residues.
• T = hydrogen bonded turn (3, 4 or 5 turn).
• E = extended strand in parallel and/or anti-parallel beta sheet conformation. Minimum length 2 residues.
• B = residue in isolated β-bridge (single pair beta sheet hydrogen bond formation).
• S = bend (the only non-hydrogen bond based assignment).
• "." = the rest of residues not belonging to any the previous classes.

DSSP computes solvent accesibility by approximating the protein surface which could be in contact with an spherical water molecule using an algorithmic called by DSSP authors "geodesic sphere integration" (see the DSSP paper for a more detailed description). Solvent accesibility values are normaliced to provide relative solvent accesibility. Normalicing values are taken from Miller et al.

RING provides the posibility of directly generating sub-networks containing only residues above or below a user defined relative solvent accesibility threshold. In this case, all the output files only contain interactions and attibues of those amino acids acomplishing the selected criteria. The by defoult threshold of 25% is the more commonly used to distinguish between buried and solvent exposed residues. This posiblity can be conbined with sub-networks containg only residues with a conservation above a certain threshold, i.g. to generate a sub-network containing only exposed amino acids with 50% conservation (see below).

Method to Compute Residue Conservation

RING produces a multiple sequence alignment in FASTA format running PSI-BLAST against the UniRef90 sequence database. In the Complex form, the user can specify the number of PSI-BLAST iterations and the minimum E-value to include sequences in the alignment. All the other PSI-BLAST parameters are the by default values. In RING, residue conservation is defined as the percentaje of sequences in the multiple sequence alignment in which an amino acid has the same identity as in the protein being studied. Highly conserved residues play an important role in the function and structure of proteins.

The multiple sequence alignment can be easily loaded and manipulated in JALVIEW, which is a useful tool to visually capture which amino acids are conserved (see output page).

RING provides the posibility of directly generating sub-networks containing only residues above a user defined conservation threshold. In this case, all the output files only contain interactions and attibues of those amino acids acomplishing the selected criteria. This posiblity can be conbined with sub-networks containg only residues with a relative solvent accesibility above a certain threshold, i.g. to generate a sub-network containing only exposed amino acids with 50% conservation (see above).

FRST Energy Calculation

RING produces an energy evaluation performed by FRST. FRST computes a per-residue energy profile of a protein structure. This profile is a composite score made of RAPDF pairwise potential, solvation potential, bakbone hydrogen bonds and torsion angle potential. For more a more detailed description see FRST reference.

TAP Energy Calculation

RING also evaluates the protein structure with TAP score. TAP evaluates the local torsion angles of a protein structure, producing a single value per residue which provides information on the likelihood that the amino acid will satisfy particular experimental quality criteria. For more a more detailed description see TAP reference.

B-factor of the C-alpha carbons

The temperature factor or B-factor can be thought of as a measure of how much an atom oscillates or vibrates around the position specified in the model. Atoms at side-chain are expected to exhibit more freedom of movement than main-chain atoms, and this movement amounts to spreading each atom over a small region of space. Diffraction is affected by this variation in atomic position, so it is realistic to assign a temperature factor to each atom and to include the B-factor among attributes available.

For this RING reports for each residue in the network, the value of the C&alpha B-factor as found in the PDB file.

From the temperature factor of the C&alpha carbons it's possible to learn what portion of the main chain has most freedom of movement and gain some insight into the dynamics of our largely static model.

Occupancy of C-alpha carbons

The occupancy of atom j is a measure of the fraction of molecules in the crystal in which atom j actually occupies the position specified in the model. It is a normalized value between 0 and 1 in which the value 1 indicates that the occupancy of all atoms in the same position is precisely identical. For example, if the two conformations occur with equal frequency, then atoms involved receive occupancies of 0.5 in each of their two possible positions.

Given that occasionally two or more distinct conformations are observed for small regions of the protein, the occupancy is included among the parameters refined. RING reports the value of occupancy of the C&alpha, as found in the PDB file, for each residue shown in the network.

In this way the user get an estimation of the frequency of alternative conformations of a given residue in the main chain of the protein, obtaining some additional information on the dynamics of the molecule.

Mutual Information between a pair of residues