POLYVIEW-3D Tutorial

Introduction

General notes

POLYVIEW-3D is a web-based tool for macromolecular structure visualization and analysis. In particular, it provides a wide array of options for automated structural and functional analysis of proteins and their complexes. This tutorial aims to describe and illustrate the available rendering options and annotation capabilities of POLYVIEW-3D. Server is available at http://polyview.cchmc.org/polyview3d.html.

By integrating the web technology with state-of-the-art software for macromolecular visualization, POLYVIEW-3D enables versatile structural and functional annotations coupled with publication quality structure rendering. In addition to static pictures, high quality animated images for electronic resources such as PowerPoint or Web-sites can be easily generated with POLYVIEW-3D as well. In particular, POLYVIEW-3D server features the PyMol program for image rendering, providing detailed and high quality presentation of macromolecular structures, with an easy to use web-based interface.

The service is platform independent and no plug-ins are required. The use of the server and its results are freely available for all users, as long as proper citations and acknowledgments are included (see below for POLYVIEW references). However, in the interest of better serving the community with limited computational resources, we reserve the right to limit the number of submissions (e.g., per day) from individual users.

We would like to thank the authors of PyMol, RasMol, DSSP and other programs that are utilized by the server (see the list below) for making them available to the community and graciously agreeing to incorporate them into a public domain server. We also gratefully acknowledge the support from NIH, University of Cincinnati College of Medicine and Cincinnati Children's Hospital Medical Center. Finally, we would like to point out that there are quite many visualization tools that are available on-line, often with complementary capabilities. One good resource to start exploring the field of macromolecular visualization is World Index of Molecular Visualization Resources.

POLYVIEW-3D focuses on annotation and visualization of protein complexes. In order to automate complex annotation tasks, it is coupled with a number of protein analysis and prediction servers, including ConSurf, CASTp, SABLE, SPPIDER, SCORPPION, as well as our previous POLYVIEW web server for protein structure visualization. Therefore, we believe that POLYVIEW-3D may become an important resource for researches and educators in structural bioinformatics, protein science and related fields. Time permitting, we will make an effort to further improve the server. Feedback regarding bugs and annoying features, as well as suggestions for improvements are highly appreciated (please use link below to contact us).

References and contact information

For citation, please use the following references:

Porollo A, Meller J: Versatile Annotation and Publication Quality Visualization of Protein Complexes Using POLYVIEW-3D, BMC Bioinformatics 2007, 8: 316.

Porollo A, Adamczak R, Meller J: POLYVIEW: A Flexible Visualization Tool for Structural and Functional Annotations of Proteins, Bioinformatics 2004, 20: 2460-2462.

POLYVIEW-3D: http://polyview.cchmc.org/polyview3d.html

Contact us and submit your feedback here.

About this tutorial

This tutorial consists of several sections that describe distinct and functionally related groups of rendering and annotation options. In order to simplify the use of the tutorial a navigation menu with expandable items is included in the left hand panel, (please click on the respective entries to toggle submenu lists).

As a general rule, each section contains a brief description of selected rendering and other options of interest, which are subsequently illustrated by examples of images generated with these options. In order to to lower the learning curve, these images are cross-linked with separate pages that contain larger versions of the images and the corresponding step-by-step instructions as to how to generate them using the server.

Most of the examples are generated using the PyMol rendering program as it produces high quality images. As an alternative, RasMol-based rendering is also available. Both programs support most of the options provided by POLYVIEW-3D with a few differences: (1) RasMol does not fully support protein surface rendering; Therefore, this option, if chosen, is automatically replaced by spherical (CPK) representation; (2) The build of PyMol (v 0.99, rev 6), which is used by POLYVIEW-3D, has a problem rendering text labels in batch mode; Therefore, the option to assign labels to selected residues is available with the RasMol rendering only; (3) RasMol and PyMol use different coordinate systems to display macromolecules, leading to problems with the initial orientation required to achieve the same structure projection. However, one can use a Jmol-based utility to set up the initial orientation of the molecule to address the latter problem (see section Initial orientation for details).

All images generated by the server can be further improved using a script for rendering provided with the corresponding picture. Thus, one can use PyMol or RasMol installed locally to improve image quality if necessary. Scripts can also serve as examples of command syntax and functionalities of the respective visualization programs, providing further hints as to how to optimize the use of the server.

Protein structure information

We start by reviewing the set of options that allows one to specify a PDB formatted file with the macromolecule of interest (e.g., protein or DNA/RNA) to be used as input for rendering.

Data source

Both the Protein Data Bank (PDB) deposited entries and the PDB formatted custom files can be submitted to the POLYVIEW server. Lines with ATOM cards are the only required data to be present in the file. For registered PDB entries, it is sufficient to enter a 4-letter code (e.g., 1a4y). In the case when both PDB code and custom PDB file name are specified simultaneously, the server will use the first rather than the latter.

For registered PDB entries, one can choose between the asymmetric unit and the biological unit if the latter is available from the Protein Quaternary Structure (PQS) server. In addition, one can indicate if ligands and/or water molecules defined in the file should be included in the image. The user can also specify the initial orientation of a macromolecule and its relative position by centering specific structural element, such as chain or residue(s) of interest.

When the file contains multiple structural models (e.g., molecular structures derived using NMR, or alternative docking models of protein complexes), one can specify the model to be rendered (by entering its number). However, this setting is discarded when the option Animate models from Animation settings is chosen. If the number of the model is not specified, then the first model is used for rendering.

Top two panels below illustrate how to select a specific model (by entering its number) from an NMR ensemble of structures, using as an example a scorpion peptide/toxin (PDB id 1acw). Second row of panels below shows an example of toggling between asymmetric and biological units, using the structure of a porin from Rh. blastica (PDB id 1bh3).

Render model 1 (Default option)	Render model 11
Asymmetric unit (Default option)	Biological unit
Click on respective image to see options used for its rendering.

Ligand and solvent data

Ligands (chemical moieties defined in HETATM sections within PDB files) and solvent (e.g., water) molecules can be optionally rendered along with the macromolecular structure. By default, ligands are shown as spheres, but can also be rendered as sticks. Water molecules are always shown as balls. In both cases, small molecules are colored using the CPK color scheme (by atom types).

Example below represents crystal structure of the Phot-LOV1 domain from Chlamydomonas reinhardtii in the dark state (PDB id 1n9l), with sulfate anion and flavin mononucleotide ligands shown using sphere and stick models, respectively.

Render ligands as spheres (Default option)	Render ligands as sticks
Show ligands and water	Hide ligands and water
Click on respective image to see options used for its rendering.

Initial orientation

There are several options to change the default projection of the structure, which is defined in the PDB file. One possibility is to specify explicitly the rotation angles (in degrees) around the three main axes (X, Y, Z). These rotations are applied to the original orientation in the order of appearance. However, the trial and error process of setting these angles manually can be tedious. To simplify it, one can use the Java-based Jmol viewer in order to select interactively the desired projection and rotation angles that are subsequently passed to the main submission form.

In many instances, it is important to highlight some particular structural elements, e.g., a mutated amino acid residue or a ligand binding pocket. However, the point of special interest typically does not correspond to the center of rendered image. In POLYVIEW-3D, one can optionally specify the center of view by entering the residue number and/or chain label. If the chain label is given without specifying a residue of interest, the chain centroid will be placed at the center of the view. Alternatively, just like the orientation, the center of focus can be specified using Jmol: clicking on residue of interest within the interactive Jmol window to assign it as a center of view will do the trick. Centering view can be also coupled with zooming.

Example below shows how the initial orientation of a protein structure can be adjusted to make its ligand binding pocket more visible. Centering and zooming options are also used in this case to highlight the ligand binding pocket better. Here, interior cavity of T4 lysozyme (PDB id 182l) is shown with two ligand specific pockets that distinguish more rigid chemical structures (benzofuran, in this case, located deeper in the cavity) and more flexible ones (2-hydroxyethyl disulfide, located in outer pocket).

Render protein and ligands (Default option)	Orient and center structure	Zoom in the pocket
Click on respective image to see options used for its rendering.

Image information

The next group of options that we discuss deals with image rendering, including the program (RasMol vs. PyMol) that is used to generate the requested picture, and the type of request, i.e., the choice between a single static image or an animation. We also describe here the preview option, which allows one to quickly refine image settings, without the often time consuming rendering of high quality pictures.

Type of request

Type of request option defines the overall type of image to be generated. Single slides represent a snapshot of the structure. Images are initially generated in the PNG format, and can be automatically converted to the TIFF format with a pre-specified DPI resolution. On the other hand, the animated images are produced in the GIF format of a size specified in the Image settings section. Each submission to the POLYVIEW-3D server opens a separate window for further processing of the resulting images.

Animated pictures are accompanied by static slides that were used as frames for the animation. These slides are available for downloading both in PNG and TIFF formats of fixed size (the maximum available size is 1000x1000 pixels). Obviously, due to the nature of multi-frame animations, time required to produce animations is significantly longer than for single slide requests. The file size and time needed to generate all frames for animation depends on angle increment, rotation angle span and the number of axes to be used for rotation (see Animation settings for details). Below, we demonstrate some options for static slide rendering and animation requests with the rocking effect, using the same protein as in ligands rendering section (PDB id 1n9l).

Single slide request (Default option)	Animation request (Rocking effect)
Click on respective image to see options used for its rendering.

Rendering program

POLYVIEW-3D utilizes at present two rendering programs, namely PyMol and RasMol. The latter is commonly used for its fast and reliable basic rendering. On the other hand, PyMol, although slower in some cases, offers a much wider array of rendering options, and can be used to produce truly impressive images, appropriate for papers, electronic presentations (e.g., PowerPoint slides) and other documents. For example, PyMol features smooth surface rendering, which can be optionally set up to be semi-transparent.

Using a protein/DNA complex as an example (PDB id 1k6o), we show the differences in rendering between these two programs with the same set of parameters. We would like to point out that, unfortunately, some options, such as the text labels that could be assigned to selected amino acid residues, are not available with PyMol rendering at present (RasMol, or post-processing of the images with graphics editors, can be used in this context as an alternative).

PyMol rendering (Default option)	RasMol rendering
Click on respective image to see options used for its rendering.

Image preview

Some complex renderings, with multiple settings to be tuned, may require multiple submissions for the same query protein, which can lead to a tedious trial and error process. In particular, animation requests usually take much time to process, which makes it difficult to refine them. To facilitate this process, POLYVIEW-3D provides the preview option that can be used to quickly generate a scaled-down version of the final image. In the case of animation requests, preview function generates and consequently presents the first frame only, reducing significantly the time needed for rendering adjustment and tuning.

Extended settings

POLYVIEW-3D provides a wide variety of options for both: molecular visualization and protein structure analysis. These different options and pertaining to them settings have been grouped into several distinct field sets that are displayed and activated in a context-dependent manner. For example, settings pertaining to animated images are only active when an animation (as opposed to a single slide) is requested, whereas settings pertaining to user provided tailored PDB files, e.g., with protein docking models in the CAPRI format, are only active when such a file is selected to be uploaded.

Currently, there are five groups of settings: (1) Image settings for defining general image parameters, such as image size in pixels, zoom level, or image background color; (2) Animation settings to specify the type of animation and some further parameters, including the angle increment for structure rotation, or the number of models to be included in the animation; (3) Residues highlighting settings for highlighting particular residues of interest, using both the color and rendering mode, e.g., spherical representation for some residues vs. cartoon for the rest of the chain; (4) Chain coloring and rendering settings to be applied to each chain; (5) Advanced structure annotation settings.

The latter group includes some of the most complex types of queries and structural analysis provided by POLYVIEW-3D. For example, it enables performing various structural and functional analyses, using both in house software, such as SPPIDER for the recognition of protein interaction interfaces, as well as several external servers, including CASTp for structural pockets identification, and ConSurf for assessment of evolutionary conservation. The results of these external resources are processed further (to a different degree) and combined with additional analysis performed by POLYVIEW-3D. One example is the ability to combine identification, mapping and prediction of protein interaction interfaces performed by SPPIDER with the analysis of putative pockets (and other topographical features) identified by CASTp. See sections below for details.

Image settings

This group of settings can be used to specify the image size, its background color, and the scaling of the structure. Images are always generated with the same horizontal and vertical dimensions, with their size defined in pixels. In the case of an animation type of request, the image size option affects the size of animated GIF only. All static slides that are used as frames for animation are available for downloading in the largest available size (1000x1000 pixels). Background color may be set up using the mouse over interactive color grid, which can be open by clicking on the Pick color hyperlink. To cancel the color selection, simply click outside the area of the color grid. The default background color, which may be changed easily as well, is set to black.

Furthermore, the zoom option may be used to bring a structural element of interest to closer view, e.g., to show in detail a ligand binding site. This option works best in conjunction with the Center of view setting, included within the Protein structure information settings group. Value of the scaling can be specified in the text field, selected from the pre-defined list, or set using the same Jmol-based utility as described in previous sections.

Images shown below are of the same protein used as an example for ligand rendering (PDB id 1n9l) in the Protein structure information section. First two images demonstrate the use of the size and background color settings, whereas the last two illustrate the use of the zooming option. The list of residues located at the ligand binding site, and rendered as blue sticks, was obtained using the option Find pockets by CASTp from the Protein structure annotation field set.

Image size 300, black background (Default option)	Image size 150, skyblue background
Zoom 100% (Default option)	Zoom 175%
Click on respective image to see options used for its rendering.

Animation settings

This section concerns the settings that can be used to create different animation effects, in particular rotation, structure fluctuation, and zooming. These settings are enabled only when animation type of request is chosen. In turn, options available within this field set are context specific and become enabled depending on the selected type of animation. The only universal option that applies to all effects is a delay between switching frames. However, the latter option has some dependence on the type of animation. In particular, if rocking effect is chosen within rotation, POLYVIEW-3D introduces increased delays (minimum of 0.5 sec and two times the pre-specified delay) at extreme positions. An increased delay is also applied in the case of the first frame in animations using structural models, and for the starting and ending zooming frames.

Rotation

This animation effect allows one to see in detail all sides of the structure of interest. It is especially useful in conjunction with opaque rendering of molecular surfaces. Options available for this type of animation include the rotation angle increment (use smaller angle for smoother rotation effect) and axes to spin the structure around. Obviously, the smaller the angle increment, the larger the number of frames to be generated and the longer the time required to finish the job.

In addition to these options, one can request the rocking effect with the corresponding angle span for it. The rocking effect takes half of the time required for full rotation effect. When rocking effect is used, the initial orientation is adjusted automatically to place the original orientation in the middle of the animation. For example, if rocking around X axis with an angle span of 90 degrees is chosen, then the initial orientation is adjusted by applying a rotation around X with dX= −45 (i.e. − 90 / 2).

Below are two examples of rotation and rocking animations. In order to reduce the image size, they had been set to have the minimum number of frames with a wide angle increment. To see smoother rotation with refined settings, click on the respective image. The structure of sucrose-specific porin (PDB id 1a0s) was used to generate these images.

Rotation around axes Y, Z	Rocking around X
Click on respective image to see options used for its rendering.

Models

When using multiple models, e.g. NMR-derived protein structures or docking models of protein complexes, one can generate a movie consisting of individual frames representing these models. By default, POLYVIEW-3D uses all models available to create this animation effect. However, it is also possible to specify how many first models should be taken into consideration.

Two images below represent animations of an ensemble of NMR conformations for the complex between palmitoyl-coenzyme A and acyl-coenzyme A binding protein (PDB id 1aca): using all available models, in the first case, and first 5 models defined in the PDB file, in the latter case.

Animation using models can be also generated using Trajectories and distortions option from Structure annotation settings. The latter always takes all models available, introducing extra delays for the first and last frames, and generating reversible motion movies.

Animate all models (Default option)	Animate first 5 models
Click on respective image to see options used for its rendering.

Zoom

In order to enlarge an active site or other structural element, one can specify the zooming for the starting and ending structure along with some increment to scale the view. The settings for the initial zooming are located in the Image settings section. In general, the smaller the increment, the smoother the effect obtained. By default, zoom-in effect is used. However, one can switch the effect to zoom-out by specifying the negative increment and reversing the starting and ending scale values. Zooming is particularly useful when coupled with center of view option, which is included in the Protein structure information section. Images below demonstrate zoom in and zoom out effect using the same protein as in the ligands rendering section (PDB id 1n9l).

Zooming in (Default option)	Zooming out
Click on respective image to see options used for its rendering.

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 Advance structure annotation for details).

Highlighting residues based on their conservation scores is similar to the option available in Chain 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 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.

Chain rendering settings

Using the set of options described in this section, one can define how each chain is to be represented. Chains can be specified individually or in groups. Several options can be set for the same chain, if combined rendering is needed. For example, sticks can be overlaid with the cartoon representation.

Individual chains settings

For each chain of a macromolecular structure it is possible to specify its rendering style and coloring scheme. In particular, a chain can be shown as cartoon representing secondary structure elements formed by the chain backbone; wireframe when all bonds including side chains are represented by sticks; CPK spacefill models represent non-hydrogen atoms as spheres of different radii depending on the atom type; smooth surface, which is available with the PyMol rendering program only, whereas use RasMol with this setting will make chain rendered in spherical representation (CPK). And finally, a chain can be set to be hidden, for example if it does not represent an interest in the context of the image.

To specify a chain to be customized, one needs to enter its label (single character) in the corresponding text field. In the case of unlabelled chains, a space or dash (minus) sign should be used. Row(s) with per-chain settings will be ignored by the server if no chain label is specified.

A number of coloring schemes are available for showing chains under different perspectives. They include simple profiles, such as hydrophobicity or temperature factors, and more complex annotations that involve additional analysis, e.g. evolutionary conservation profiles or mapping interacting residues from homologous proteins to the query chain.

Currently, the following coloring schemes are available:

1. By custom color − allows one to pick a color from the color grid, and to apply it to the corresponding chain. In addition, the selected color is also shown in HTML format for reference.
2. By atom type − can be used to color the atoms of the backbone and side chains according to their properties.
3. By B-factor − shows temperature factors optionally included in PDB files to estimate the extent of thermal fluctuations and identify rigid, as well as relatively flexible regions of the structure. Note that each rendering program has its own coloring scheme for representing temperature factors. Moreover, many programs or web-servers use B-factor field to encode some other information, e.g., conservation scores or class assignments. POLYVIEW-3D accepts prediction results in such a way from two web-servers, SPPIDER and ConSurf (see legends below).
4. By conservation − invokes a PsiBLAST job to calculate a (pseudo-) multiple sequence alignment and the corresponding position specific conservation scores, as defined in the Residue highlighting settings section. All residues within the chain are colored according to their conservation, see color scale below.
5. By interaction − performs an automatic search for close sequence homologs of the query chain deposited in PDB, and involved in different types of interactions. Interacting residues with interchain contacts are identified and mapped to the corresponding residues of the query chain. Interacting sites are colored according to the type of interaction (see legend below), whereas the rest of the chain is white. Note, that due to mapping from multiple complexes, the same residue may be found to interact with another protein chain or a DNA molecule, depending on the complex.
6. By hydrophilicity − colors residues by the hydrophobicity, polarity, charge and combination of these properties, see amino acid assignments below.
7. By acidity − discriminates basic and acidic residues, other amino acids are colored in white, see assignments below.

To add or delete rows of per-chain settings, use the corresponding + and − buttons on the right hand side of the option text fields.

Images below demonstrate several alternative ways of rendering protein chains along with different color schemes applied. As an example, a high affinity protein complex between human placental RNase inhibitor (hRI) and vessel-inducing human angiogenin (Ang) is shown under various perspectives (PDB id 1a4y). Panel in the middle might help explaining the high binding affinity due to extended electrostatic interactions.

Rendering: cartoon (hRI), wireframe and CPK (Ang)	Surface rendering (hRI) and coloring by hydrophilicity (hRI) and acidity (Ang)	Coloring by conservation (hRI) and known interactions (Ang)
Click on respective image to see options used for its rendering.

Other chains settings

All chains not specified in individual chain settings fall into the group called rest chains. And the same rendering settings as well as coloring schemes defined in previous section can be applied to them as a group with a few differences. Analog of By custom color option is called All in same color and allows one to shade the remaining chains down from those of special interest using the same color. In the case of structures with multiple chains, it is convenient to select coloring scheme By chain that makes rendering programs automatically assign different colors to each chain. Note that PyMol and RasMol apply their own conventions on colors when performing this function.

Caution should be exerted when applying coloring schemes By conservation and By interaction to the group rest chains. In the case of multichain structures, it may require a considerably long period of time to fully process this kind of request.

Protein structure annotation settings

There are two general groups of settings within this options set. The first group invokes automatic requests to other web-servers and databases to obtain additional structural annotations. The second group uses precomputed results that were previously received (and possibly processed) from other resources. In both cases, the resulting data is further processed by the POLYVIEW-3D server in order to yield 3D images of the macromolecular structures overlaid with the requested annotations, as described below.

Finding pockets using CASTp

Invoking this option results in a request sent automatically to the CASTp server in order to perform a search for structural pockets within a given query protein. Such identified pockets can be filtered by their area or volume. These putative pockets are also automatically colored according to the CASTp color convention and characterized in tabular form in terms of both atoms and residues they comprise. Pockets can also be optionally overlaid with the prediction of interacting sites automatically performed using the SPPIDER server. Pockets overlapping with predicted interfaces may represent interesting targets for drug design and docking simulations.

Below is an example of a request to find pockets using CASTp, with pocket area cutoff of 100Ų. The structure of purine nucleoside phosphorylase in transition state was taken as a query structure (PDB id 1b8o).

Pockets larger or equal 100Å2
Click on respective image to see options used for its rendering.

Generated image is accompanied by annotation table with the lists of residues located at each of the pockets.

Determination of domains using Pfam

When this option is selected, POLYVIEW-3D performs a sequence homology search using BLAST against local version of Pfam database. Amino acid sequence used for query is derived from ATOM section of the PDB file. If significant sequence homology (E-value ≤ 0.001 AND sequence identity ≥ 70%) is found to one or more domains, they are mapped to the queried structure using distinct colors for each domain. The same structure as in previous example was used as a query (PDB id 1b8o) in the image below.

Domains from Pfam
Click on respective image to see options used for its rendering.

Generated image is accompanied by annotation table with the lists of residues found to belong to different domains.

Determination of putative TM segments using PDBTM

To process the request with this option chosen, POLYVIEW-3D makes search in local copy of the PDBTM database of putative trans-membrane segments. Depending on the source of the protein structure the server either seeks the data by the PDB code or performs a sequence homology search using BLAST. In the latter case, amino acid sequence used for query is derived from ATOM section of the PDB file. If significant sequence homology (E-value ≤ 0.001 AND sequence identity ≥ 70%) is found to one or more proteins, the best matching homolog is taken. Images below demonstrate the membrane spanning segments annotation as determined in PDBTM using the TMDET automated algorithm (for details, please refer to the original papers). The structure of Catalytic Core (Subunits I and II) of Cytochrome c oxidase from Rhodobacter sphaeroides serves as an example of alpha-helical TM protein (PDB id 2gsm), whereas the structure of the sucrose-specific porin ScrY (PDB id 1a0s) from Salmonella typhimurium represents beta-barrel TM proteins.

TM segments from PDBTM Alpha helical	TM segments from PDBTM Beta barrel
Click on respective image to see options used for its rendering.

Generated image is accompanied by annotation table with the lists of residues computationally determined to be membrane spanning segments.

Mapping interacting sites from PDB complexes using SCORPPION

With this option, one can retrieve information about all interacting sites found among close sequence homologs deposited in PDB. Search is automatically performed using the SCORPPION server. Interacting sites are the residues in contact with other protein chains, DNA or RNA, or ligands. Different types of interactions, as well as their combinations are colored distinctly. Interacting sites can be optionally contrasted with protein interfaces predicted using SPPIDER.

Below is an example of mapping different types of interactions from close sequence homologs to a query structure of the transcription factor CSL (PDB id 1ttu, chain A).

Interfaces by SCORPPION
Click on respective image to see options used for its rendering.

Generated image is accompanied by annotation table with the lists of residues found to have different types of contacts.

Predicting protein interface using SPPIDER

This setting belongs to the second group of options that utilize pre-computed results, which were received prior to submitting a query to POLYVIEW-3D. The SPPIDER server optionally generates a PDB file with temperature factor fields modified according to the probability of that residue being involved in protein-protein interactions. These files can be submitted to the POLYVIEW-3D server directly, as custom PDB files with chains of interest to be colored using By B-factors color scheme (see Chain rendering settings).

Examples below illustrate the use of POLYVIEW-3D in this case, with interacting sites found in the CSL transcription factor (PDB id 1ttu, chain A) using SPPIDER. The predictions are encoded using both classification and regression approach (with a binary class assignment in the first case, and a probability of being within an interaction interface in the latter case, respectively), and incorporated in the corresponding B-factor fields. Color scheme applied is By B-factor and described in Chain rendering settings section. PDB files modified by the SPPIDER server and used for the images are also available for downloading (classification-based and regression-based prediction).

Interfaces by SPPIDER as classification	Interfaces by SPPIDER as regression
Click on respective image to see options used for its rendering.

Generated images go along with annotations where residues are grouped by either classes or probability bins (see corresponding tables below).

Finding functional regions using ConSurf

The ConSurf server ranks residues according to their relative conservation scores that can be used for the identification of putative functional regions within a given structure. The server also modifies the original PDB file to encode conservation scores by replacing B-factor fields. This kind of file can be directly submitted to POLYVIEW-3D. By specifying chain of interest to be colored by B-factors, one can obtain high-quality images of ConSurf results with its original coloring scheme.

Below is an example of ConSurf output, with conservation scores encoded as B-factors and residues colored using the original color scheme. The same protein was used as in the previous section (PDB id 1ttu, chain A). Color scheme applied is By B-factor and described in Chain rendering settings section. PDB file modified by the ConSurf server and used for the image is also available for download.

Functional regions by ConSurf
Click on the image to see options used for its rendering.

Generated image is accompanied by annotation table with the lists of residues found to have different types of contacts.

Analysis of docking models from CAPRI

POLYVIEW-3D provides an option for analyzing the docking models in the CAPRI format obtained by different programs or web-servers for protein-protein docking. In addition to visualization of docking models, POLYVIEW-3D performs their analysis and assessment. In particular, a custom PDB file with multiple models of a protein complex in the CAPRI format (e.g., generated by the ClusPro server), when submitted to POLYVIEW-3D, triggers SPPIDER predictions in the background for both chains that are docked. Unbound structures of these chains are used to predict putative interaction interfaces, which are then compared with interfaces observed in each model. The fraction of residues within the interface in a given model that overlaps with SPPIDER predictions (averaged over both chains) provides a simple score to rank these models. In addition, the surface area, average hydrophobicity, and evolutionary conservation for each interface within these models are computed to provide a basis for further analysis and visualization. Models can be then re-ranked according to these values and sent to refine image rendering.

The table below includes the output of POLYVIEW-3D assessment of docking models for the system used as CAPRI target #9, and described in more detail in Example 2 of the Advanced examples section. The two chains were submitted to the ClusPro server, in order to obtain 10 best ranking models of the protein complex (download results). In addition, the option for models assessment by overlapping with protein interfaces predicted by SPPIDER has been specified. Note that table rows (and thus different models) can be easily ordered (re-sorted) according to measures included in columns three to five.

Analysis of docking models (Click on the header of column to re-sort models)

Model	Image	(1) ASA, Å2	(2) HP index	(3) Overlap, %	Residues at interface
1		2436	1.04	54.03	Chain: A (Overlap=58.06%, Sum(ASA)=1246 Å2, Mean(HP)=1.06±0.72) Red: 2 3 5 6 7 9 ... Blue: 21 37 63 112 ... Yellow: 1 4 8 12 13 14 ... Chain: B (Overlap=50.00%, Sum(ASA)=1190 Å2, Mean(HP)=1.01±0.72) Red: 2 3 5 6 7 9 ... Blue: 21 37 38 46 ... Yellow: 1 4 8 12 13 14 ...
2		2150	0.72	42.04	Chain: A (Overlap=37.93%, Sum(ASA)=1081 Å2, Mean(HP)=0.68±0.67) Red: 1 2 3 6 41 ... Blue: 34 36 37 38 ... Yellow: 4 5 7 8 9 10 ... Chain: B (Overlap=46.15%, Sum(ASA)=1069 Å2, Mean(HP)=0.75±0.67) Red: 1 2 3 6 41 ... Blue: 34 36 37 38 ... Yellow: 4 5 7 8 9 10 ...
3		2438	0.95	27.25	Chain: A (Overlap=28.57%, Sum(ASA)=1218 Å2, Mean(HP)=0.95±0.73) Red: 5 8 9 12 49 53 61 134 Blue: 59 62 63 65 ... Yellow: 1 2 3 4 6 7 ... Chain: B (Overlap=25.93%, Sum(ASA)=1220 Å2, Mean(HP)=0.95±0.74) Red: 5 8 9 12 49 53 134 Blue: 59 61 62 63 65 ... Yellow: 1 2 3 4 6 7 ...
4		2091	0.96	47.22	Chain: A (Overlap=44.44%, Sum(ASA)=1050 Å2, Mean(HP)=0.98±0.67) Red: 3 7 10 11 15 17 18 40 Blue: 21 25 28 29 32 ... Yellow: 1 2 4 5 6 8 9 ... Chain: B (Overlap=50.00%, Sum(ASA)=1041 Å2, Mean(HP)=0.95±0.67) Red: 3 7 10 11 15 17 ... Blue: 21 25 28 29 32 ... Yellow: 1 2 4 5 6 8 9 ...
5		2374	1.02	16.16	Chain: A (Overlap=18.52%, Sum(ASA)=1176 Å2, Mean(HP)=1.00±0.74) Red: 53 61 134 137 139 Blue: 59 62 63 65 66 ... Yellow: 1 2 3 4 5 6 7 8 ... Chain: B (Overlap=13.79%, Sum(ASA)=1198 Å2, Mean(HP)=1.03±0.73) Red: 53 134 137 139 Blue: 59 61 62 63 65 66 ... Yellow: 1 2 3 4 5 6 7 8 ...
6		2479	0.83	45.21	Chain: A (Overlap=48.48%, Sum(ASA)=1253 Å2, Mean(HP)=0.88±0.69) Red: 1 2 3 6 7 40 41 ... Blue: 21 28 31 32 33 ... Yellow: 4 5 8 9 10 11 ... Chain: B (Overlap=41.94%, Sum(ASA)=1226 Å2, Mean(HP)=0.79±0.69) Red: 1 2 3 6 10 40 146 ... Blue: 31 32 33 34 36 ... Yellow: 4 5 7 8 9 11 12 ...
7		1276	0.79	46.05	Chain: A (Overlap=50.00%, Sum(ASA)=634 Å2, Mean(HP)=0.80±0.69) Red: 1 2 3 5 6 10 41 ... Blue: 36 161 163 174 ... Yellow: 4 7 8 9 11 12 ... Chain: B (Overlap=42.11%, Sum(ASA)=642 Å2, Mean(HP)=0.77±0.70) Red: 1 2 3 5 6 10 170 194 Blue: 36 161 162 163 ... Yellow: 4 7 8 9 11 12 13 ...
8		2305	1.02	24.96	Chain: A (Overlap=25.93%, Sum(ASA)=1165 Å2, Mean(HP)=1.00±0.69) Red: 6 53 56 58 61 134 139 Blue: 59 62 63 64 66 67 ... Yellow: 1 2 3 4 5 7 8 9 ... Chain: B (Overlap=24.00%, Sum(ASA)=1140 Å2, Mean(HP)=1.04±0.69) Red: 6 53 56 58 134 139 Blue: 59 61 63 64 66 67 ... Yellow: 1 2 3 4 5 7 8 9 ...
9		2081	0.89	45.75	Chain: A (Overlap=44.44%, Sum(ASA)=1056 Å2, Mean(HP)=0.86±0.71) Red: 144 147 151 165 ... Blue: 150 154 158 162 ... Yellow: 1 2 3 4 5 6 7 8 ... Chain: B (Overlap=47.06%, Sum(ASA)=1025 Å2, Mean(HP)=0.91±0.70) Red: 144 147 151 165 ... Blue: 150 154 158 162 ... Yellow: 1 2 3 4 5 6 7 8 ...