POLYVIEW-3D Tutorial

Part 4. Residues highlighting settings

This set of options allows one to specify individual amino acid residues to be highlighted by either color or different type of rendering compared to the rest of the structure. Residues to be highlighted can also be selected automatically, using several predefined annotation options that allow one to identify sites of interest, e.g., evolutionarily conserved functional hotspots, protein-protein interaction interfaces, etc.

Individual highlighting

POLYVIEW-3D provides a possibility to highlight a list of residues that represent, e.g., some structurally or functionally relevant group. This can be achieved by using a different color, different rendering style (e.g., stick vs. cartoon model), or by the combination of those. The list of residues to be highlighted may be specified using a text input field included in the Highlighting settings (shown when the Residue Highlighting option is checked). The format for custom highlighting is as follows: [Chain:]Residue:{Color|Rendering}, where Chain is an optional chain label of the chain a residue of interest belongs to; Residue is the number of the residue (along with an optional insertion code, e.g., 10A, 10B, 10C if three alternative definitions of residue number 10 are provided in the PDB file); Color is represented by a single letter from a pre-defined set of possible values:

For example, string A:5-10:r,A:27:bs,B:101-103:f would result in rendering residues 5 through 10 of chain A in red; residue 27 of the same chain in blue with its side chain shown using sticks; and all atoms of residues 101 through 103 shown as spheres.

Please note that if at least one residue is specified to be rendered with transparent surface, all other surfaces will also be shown as transparent, as a result of a global setting in PyMol, which is applied to all instances of surface representation.

If a few residues are to be highlighted, one can enter the list (and the corresponding selection string) manually. However, for more complex annotations, we recommend a list conversion tool, which is available via Converter link located next to highlighting option. This tool allows one to process residue list in two ways, converting a simple selection string to the POLYVIEW format, while taking into account color and 3D rendering settings to be applied, and in the opposite way, allowing one to generate a plain list of residues from the one formatted automatically by the server, e.g., when performing various structure analyses. In both cases, these strings can be pasted to or copied from the respective text fields, faciltating the task performed. Note that adjacent numbers of residues can be replaced by their range defined by hyphen (e.g., 10-12:v instead of 10:v,11:v,12:v).

Examples below illustrate custom residue highlighting, using different colors and rendering styles. Protein in the left panel is the potassium channel KirBac1.1 in the closed state (PDB id 1p7b), with residues in transmembrane (TM) regions colored in yellow and rendered with side chains. For contrast, all other chains are colored in light blue. TM regions are taken from the PDBTM database. Panel in the middle represents a ligand binding pocket of the Phot-LOV1 domain (PDB id 1n9l), as identified by the CASTp server, colored in blue and rendered with side chains. The right panel shows two histidines (His²⁰⁷ and His²⁶⁹) as important phosphorylation sites that regulate the dimerization of LicT regulatory domain (LicT-PRD, PDB id 1tlv). The residues of interest are colored in red and rendered as spheres with side chain atoms.

Membrane spanning regions	Ligand binding pocket	Phosphorylation sites
Click on respective image to see options used for its rendering.

Highlighting by interaction

The POLYVIEW server provides an option for automatic protein interface recognition and highlighting. This option is enabled when either a protein complex is directly submitted or a PDB entry with multiple chains in its asymmetric or biological unit, as defined by PQS, is requested. Residues that are found to be within the interaction interface can be optionally highlighted using one of the following options: colored in red, side chains rendered as sticks, or represented as spheres. The respective per-chain list of residues will appear in the resulting annotation page, next to the image of the complex.

In order to define protein interaction interfaces, the change in solvent accessibility area (SA) upon complex formation is calculated using the DSSP program for each residue. For that purpose, the solvent exposed surface area of each residue is computed twice: using the isolated chain considered as unbound structure, and the protein complex that represents the bound state:

dSA = SA(Unbound) − SA(Bound)

Residues with dSA of more than 4% of the maximum possible surface exposed area for a given type of amino acid, and more than 5 Ų, are assigned as interacting sites (see SPPIDER paper for the justification of this particular choice). In order to use non-default settings one has to use the SPPIDER server directly, and then refine the image in POLYVIEW-3D.

An example shown below illustrates the automatic recognition of protein interface between alpha and beta subunits of chicken sarcomeric capping protein CapZ (PDB id 1izn) based on the structure of its biological unit.

Interacting sites with side chains	Interface in red
Click on respective image to see options used for its rendering.

Highlighting by conservation

Another way of highlighting residues provided by POLYVIEW-3D is to calculate their corresponding conservation scores based on multiple sequence alignment (MSA). By setting different thresholds, one can obtain a list of either highly conserved residues or the most variable ones, thus identifying potentially interesting and functionally relevant regions of the protein. Highlighting those residues can be performed in the same manner as described in previous section.

In order to calculate conservation scores, POLYVIEW-3D server uses frequencies of amino acids occurred at a given position obtained using MSA against NCBI nr protein database. Based on these frequencies, the amino acid entropies are computed as follows:

S = −∑_i (F_i * ln(F_i)) / ln(N)

where N is the number of amino acids (20), and F_i is the frequency of i-th amino acid at a given position. The resulting conservation score C is computed as:

C = {integer((1 − S)*10), 9 if C > 9}

Thus, C=0 represents the most variable positions, whereas C=9 the most conserved ones.

Note that this definition is different to that used by the ConSurf server, which provides an alternative assessment of evolutionary conservation and mapping of putative functional hot spots (please refer to ConSurf documentation for details). However, POLYVIEW-3D also accepts PDB files generated by the ConSurf server with B-factors modified according to its conservation scale and produces the images with the original ConSurf coloring scheme (see section Advanced structure annotation for details).

Highlighting residues based on their conservation scores is similar to the option available in Chains coloring and rendering field set. However, instead of coloring the whole chain, only selected residues are highlighted based on the threshold for the conservation score that is applied.

Images below demonstrate this option, using the same protein complex as in previous section (PDB id 1izn). As can be seen from these images, highly conserved residues are mostly located at the protein interface, whereas the rest of the structure is rather variable in this case.

Conservation score > 6	Conservation score < 3
Click on respective image to see options used for its rendering.

When RasMol is used as a rendering program (see section Image information), one can optionally request to show text labels for highlighted residues. However, we suggest that labels should be used for reference only, e.g., when locating specific residues. For the final image, it is recommended to add labels manually by using some graphics editor.

Below are some examples of rendering with labels for highlighted residues, using again the same proteins as above (PDB ids 1n9l, 1izn, 1tlv).

Ligand binding pocket	Highly conserved spots	Phosphorylation sites
Click on respective image to see options used for its rendering.

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