| © 1997-2017 Luc Moulinier |
| Complex Systems and Translational Bioinformatics, |
| ICube laboratory |
| 11, rue Humann |
| 67 000 Strasbourg |
| FRANCE |
A protein family can be defined as a group of proteins related through evolution that share similar 3-D structures and functions, leading usually to sequence conservation. The concept of protein family has been established in the 70's where few protein sequences and structures were known and most of them were small and constituted of a single domain. Since then, the massive increase of protein structures and sequences led to more subtle definitions, like super-family or sub-family organizations.
Studying a protein family consists now in characterizing all features that specify the family, not only at a structural, functional, phylogenetic or residue conservation level, but also by using all related information available in various databases. Indeed, more and more information is available for all aspects of protein characterization, which mainly arises from high throughput technologies of the post-genomic era such as genomics, proteomics, interactomics or transcriptomics. Handling such information remains a difficult task because of its heterogeneity (3D structure, transcriptional level in time and space, ...) and deals with several levels of detail, ranging from very local data like point mutations up to large scale data like cellular localization, domain or macromolecular complexes organization or interaction. As a consequence, a new member of a protein family is then surrounded by information that can be assigned to it. Such data harvesting and assignation has been implemented in the Macsims software [10], which integrates and propagates heterogeneous information in the environment of the multiple sequence alignment of a protein family. A remaining problem resides in the analysis and the visualization of this information.
Ordalie (ORDered ALignment Information Explorer) is an interactive tool designed for the exploration of the informational content of a multiple sequence alignment into a hierarchical manner, and within different contexts, such as phylogeny or 3D structure.
The Ordalie philosophy (see fig. 1) resides in its ability to make a concomitent multi-scale analysis along three axes : the aminoacids sequence axis, the taxa axis, and the contexts axis.
The information running along the aminoacid sequence (horizontal axis) can be seen according to several scales:
Another analysis axis resides in the way the different taxa present in the alignment are handled. The study can be done at a global level (all taxa) to characterize the whole family through different features, such as conserved motifs or key signature, it can also be done on a particular taxon to identify and specify point mutation positions, or at an intermediary level to study the features allowing sub-family identification, such as differentially conserved residues between the sub-family and the other taxa.
As a third analysis axis, Ordalie embeds tools allowing different analysis contexts: residue conservation computation, phylogenetic tree computation and rendering, external features mapping, a 3D structure viewer, etc .... All analyses can be done in a structural context, as all available features can be mapped and compared on the available 3D structures present in the alignment.
For a given alignment loaded in Ordalie, it is easy to understand that many different instances of this same alignment may exist. One instance could have a given set of sequence clusters with a given sequence conservation computation, and an other instance could have an other set of clusters, in order to estimate different hypotheses. These instances are called ``snapshots'' in Ordalie and can be annotated, saved and retrieved at any time. This is made possible thanks to the database embedded in Ordalie.
As a conclusion of this short introduction, the strength of Ordalie for a protein family analysis resides in the cross-comparison of all information seen in different contexts and at different scales. By adjusting the coarseness of the scale (all taxa, a subgroup of taxa, or a taxon alone for example), the outcoming information will help in deciphering different aspects of the sequence - structure - function - evolution relationships for the protein family under study.
Ordalie is dedicated to the analysis of protein multiple sequence alignments. Although it can read DNA/RNA alignments, most of its functionalities will be disabled. Ordalie can still be used to view or edit such alignments.
Ordalie can read and write the fasta, MSF, RSF, ClustalW, Macsim/XML and ORD (Ordalie file format) file formats. Once the alignment is loaded, Ordalie tries to recognize if the sequences names are UniProt, RefSeq, or Protein Data Bank (PDB) accessions names. If a sequence name is prefixed by a database identifier (for example, sw for swissprot, gi for Gene Identifier, pdb for PDB) the prefix will be removed by default. Thus, the sequence name >sw|P12345 will appear as P12345 in Ordalie. The list of recognized bank prefixes and their separator can be changed through the 'Preferences' menu item.
If sequence names are proper databases accession, Ordalie can then fetch information on these databases.
All the different menus are described in detail in section 5 of this manual. In short, the ``File'' menu manages input/output files, as well as adding sequences or printing. The ``View'' menu controls the appearence of the user interface. It contains options to toggle on or off parts of the main window, to change the font size, or to toggle the full-screen mode. The ``Sequence'' menu allows to change the sequence names, browse, edit or retrieve sequence information, search for sequence motif, compute sequences identity, access the vectorial representation of protein. The ``Alignment'' menu gives access to all tools linked to the alignment: creation of a Macsim, alignment editor, clustering, phylogenetic tree, features editor, ... The ``Structure'' menu is dedicated to the structural analysis of the alignment if any sequence corresponding to a 3D structure is present. The menu gives access to a structure superposition module, the 3D viewer, a secondary structure coloring scheme according to sequence conservation, and allows to save PDB files.
Below the menus, the icon bar gives a direct access to some of the most useful menu items. When the mouse pointer is above a button, a small message box describing the button's action appears.
As previously mentioned, working with an alignment may lead to several trials in terms of sequence clustering or even amino acid alignments. A trial can be saved as a snapshot of the loaded alignment. A given snapshot can also contain a different set of sequences than the original loaded alignment in case of deletion or addition of sequences.
From left to right, the combobox allows to select a given snapshot. The ``Annotation'' button shows or hides the annotation of the current snapshot if they exist. Annotations are created through the ``Annotate Alignment'' item in the Alignment menu. The ``View Zone'' button toggles the zone used to make the clustering of the given snapshot if it has been clustered. The ``Info'' button pops up a windows displaying the information relative the the snapshot. These information are sought when creating the snapshot. The ``Reset'' button will reload the current snapshot which will erase all changes made so far. The ``Overwrite'' button saves the current changes to the current snapshot while the ``New'' button creates a new snapshot.
The sequence names are displayed on the left part of the frame, the amino acid sequences on the right part.
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The sequence names highlighted in red correspond to PDB sequences. If there is information associated to a given sequence (present in Macsim/XML, ORD files or retrieved on-line, see 5.4.4) a yellow message window containing a description of the current sequence appears above the sequence pointed by the mouse pointer. A right-click (mouse button-3) on a given sequence name displays a more detailed message window containing the accession, the bank ID, the organism, the length and the description of the sequence.
Below the sequence names, an entry box allows the user to search a sequence by its name, or part of its name. After hitting <Return> the first sequence found will be displayed as the top sequence in the window.
The right part of the frame contains the alignment itself (amino acids sequences), the ruler, indicating the position of the column, the horizontal and vertical scrollbars and the position counter. Any mouse motion above the amino acid sequences will update the position counter that shows two positions for the residue below the mouse pointer: the 'seq' position is the position of the residue inside its sequence, the 'gen' position corresponds to the position of that residue inside the alignment.
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When a given feature is displayed, moving the mouse over the feature will display the note associated with it, for example, in the case of a PFAM domain, the description of the domain will be shown. If there are severeal features superposed, the first description corresponds to the top feature.
This frame is not shown by default. When residue conservation has been computed, a score is assigned to each column of the alignment and groups. The Scores frame shows these normalized scores (between 0 and 100) for each column, the colour of the score line corresponding to the group color, the black line corresponding to the whole alignment.
It is also possible to jump from position to position using the numeric keypad and the left and right arrows. For example, by typing '200' + <Right Arrow> key, the window will go 200 positions to the right. Similarly, typing '500' + <Left Arrow> key will scroll the alignment 500 positions to the left.
The Control Panel is at the bottom of the main window. When available, this frame contains buttons corresponding to the available features of the current alignment, one button for one feature. Pressing a button will render the button red, and display the feature on the alignment.
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When changing tool, the content of the Control panel will change according to the tool. The content of the Control panel will be described in each tool section.
Features are a central concept in Ordalie. A Feature can be defined as a characteristic attached to a sequence, a group of sequences or to the global alignment. A sequence / group / alignment feature can contain several items (for example, a sequence feature can contain several PFAM domains). One of the strength of Ordalie is its ability to investigate these features in different contexts, for example in the structural context of the protein.
Features are imported into Ordalie through the Macsims program XML output file [10], or using a dedicated feature file format (see section 6.4), or manually defined using the 'Feature tool'. This tool allows feature creation, edition or deletion (see section 3.7).
In Ordalie, a feature is defined by the sequence(s) it applies to, a start and stop position, a color, an associated score, a note and a coordinates system ("global" for alignment position or "local" for sequence relative position).
Ordalie can display, modify or create new features or items of features. Features can usually be displayed and selected in all modes (a special mode, the 'Feature mode' is dedicated to features editing).
Ordalie is arranged around tools. To achieve an action in Ordalie, one should enter the corresponding tool. For example, editing an alignment requires to go into the 'Editor' tool, computing a phylogenetic tree to enter the 'Tree' tool, etc ... All tools will be described in detail in section 3.
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In this manual, the mouse left, middle and right buttons will be designed as <B1> or <Button-1>, <B2> or <Button-2>, <B3> or <Button-3> respectively. Any words enclosed by '<' and '>' refer to the corresponding keyboard key.
In all tools, sequence names and sequence range selections are always done the same way.
Sequences can be selected by left-clicking on their names. The selection mechanism obeys standard rules :
A Cut/Copy/Paste mechanism is available at any time, and allows the user to duplicate, remove or change sequence order. Cut/Copy/Paste are available through the 'Edit' menu or by hitting <Control-x>, <Control-c>, and <Control-v> keys respectively.
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Several zones can be defined one after the other, either by left/right clicks and/or feature selection.
In order to manage snapshots, features, 3D structures, etc... Ordalie internally embeds a SQLite database [3]. This database is lightweight, and can easily be copied or moved around. The Ordalie file format (.ord extension) is in fact the SQLite database itself.
The scheme of the database can be found in Appendix 6.2.
In short, the database contains :
As Ordalie files (the SQLite database) contain all the information, it should be prefered as being the default working format.
There are mainly two types of tools in Ordalie: tools that query or add information (Search, Identity, Tree, Conservation, ...) and tools that change the current snapshot (Editor, Cluster, Feature etc ...). There are shortcuts that allow to enter a tool using the keyboard :
| Key | Tool |
|---|---|
| <Shift + A>, <A> | Annotation |
| <Shift + C>, <C> | Conservation |
| <Shift + E>, <E> | Editor |
| <Shift + F>, <F> | Feature tool |
| <Shift + G>, <G> | Clustering |
| <Shift + I>, <I> | Identity |
| <Shift + M>, <M> | Search motif |
| <Shift + S>, <S> | Superpose |
| <Shift + T>, <T> | Tree building |
A fruitful multiple sequence alignment exploitation ensuring a high quality of data usage by tools and feature mapping is directly dependent on the accuracy of the alignment. Although the research on algorithms dedicated to aligning sequences is still intensive and the outcoming softwares are more and more accurate, the need of manual MSA inspection, curation and editing is still necessary. This is the reason why Ordalie integrates a high performance sequence editor. It is written in C to ensure speed and fluidity, and is inspired by SeqLab [11], the editor GCG Wisconsin Package.
Entering the Editor tool will first clear the sequence from any displayed feature and colour the sequences according to physico-chemical properties. The default colouring scheme is :
(The default scheme can be changed inside the 'Edit -> Preferences' menu item).
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At the bottom of the Ordalie window, the Control panel will display the following buttons from left to right.
Clears any current sequence selection.
This will group the selected sequences. The names of the grouped sequences will be coloured in a different colour, a unique colour for a given group. If one or more sequences already belong to a group, the user should decide through a dialog box, if the sequences should be merged with an existing group, or if a new group should be created.
Grouped sequences will behave as a single sequence.
The selected sequences will be removed from any group. If a group consists of only one sequence, the group is automatically destroyed.
By default, only gaps ('.') can be deleted or inserted, it is not allowed to insert/delete amino acids. Unlocking the sequences allows the insertion/deletion of residues.
Ordalie runs through all alignment columns and removes those containing only gaps. A 'Rem. Col. Gap.' is automatically done when leaving the 'Editor' mode.
This creates a TFA file copy of the current alignment under edition. The user is asked for a file name the first time, and successive 'Save' will use this file name to output the alignment.
Leaves the 'Editor' with all the changes made. A dialog box will asked the user if the current alignment should be overwritten, or if a new alignment should be created with the current changes.
Leaves the 'Editor' and restore the original alignment.
Clicking inside the alignment frame will show a yellow blinking cursor. The following actions are then available :
In order to speed up editing, pressing keypad or keyboard digits [0-9] stores the number in a buffer, i.e. pressing '1' then '2' will store number 12 in the buffer. Pressing then the <Left> arrow will move the cursor 12 characters to the left, and will empty the buffer. All the actions previously described can take advantage of this mechanism.
The first method consists in adding/deleting empty lines, called 'separators' hereafter, between sequence names. Separators can be added using the corresponding icon on the Icons bar, or through the 'Add separator' item of the 'Alignment' menu and deleted using the corresponding icon or item menu. The separator is added just below the selected sequence. Similarly, the separator just below the selected sequence will be removed if requested. Sequences enclosed by separators constitute then a new group. The 'Remove all separators' item of the 'Alignment' menu removes all groups in the current snapshot.
The 'Clustering' tool allows the creation of clusters (or groups) based on numerical criterions characterizing the sequences to be clustered. The computation can be done using all or part of the sequence as well as all or part of the snapshot columns. The user chooses one or more numerical criterions as the basis for the computation and a clustering algorithm. The computation can then be launched and the newly created sequence clusters are automatically displayed in the main window.
At present, the available criterions are :
Ordalie clusters and automatically defines the number of groups. The clustering algorithms along with the algorithms that define the number of clusters are taken from the Cluspack package. The available methods are :
If no sequence names are selected, the clustering will use ALL sequences. If some sequences are selected (more than 3), then the clustering will only apply to these selected sequences. The remaining ones will be kept as a separated group.
The pull-down menu allows the selection of the criterias to be used for the computation. Several criterias can be selected at the same time.
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The 'Method' pull-down menu allows to choose the algorithm to be used for clustering computation.
The ``Compute'' button will launch the computation. The newly computed sequence clusters are directly displayed in the main ordalie window.
The 'Reset' button will erase any clustering done so far and show the original clustering if any. The 'No Clusters' buttons removes all groups and leaves all the sequences as a single group.
The ``Clusters Names'' button will pop up a window allowing to give a name to each cluster. this cluster name may be used in subsequent analysis to identify the clusters, like in the ``Tree'' display, or the ``Barcode'' tool.
The 'Save' button will leave the clustering tool and the current clustering will be saved. The user is prompted whether to overwrite the current snapshot or to create a new one. The 'Return' button leaves the Clustering tool and displays the snapshot in its original state.
This tool is used to query information on identity percentages between sequences.
The 'Compute' button calculates the identity percentage between selected sequences for the selected residue range. A summary of the computation is logged. The selection of two sequences for which the identity percentage is desired is done with the following two comboboxes. The identity percentage and the length of the two ungapped sequences is then given.
The 'Summary' button will make a window appear that will give for the whole sequence and for each group :
The 'Return' button will leave the Identity tool.
This tool allows the user to search for a particular sequence motif inside the alignment.
The syntax of the search pattern follows the rules of the FindPatterns program of the GCG Wisconsin Package [15]. The following subsections are adapted from the FindPatterns documentation.
The search pattern can include any legal sequence character, and also include several non-sequence characters, which are used to specify 'OR' matching, 'NOT' matching, 'begin' and 'end' constraints, and repeat counts. For instance, the pattern GASTE(X){20,30}FTG means searching GASTE, followed by 20 to 30 of any amino acid, followed by FTG. Following is an explanation of the syntax for pattern specification.
Sometimes, it is posssible to leave out part of an expression. If braces appear without preceding parentheses, the numbers in the braces define the number of repeats for the immediately preceding symbol. One or both of the numbers within the braces may be missing. For instance, both the pattern GASG{2,}F and the pattern GASG{2}F mean GAS, followed by G repeated from 2 to 350,000 times, followed by F; the pattern GASG{}F means GAS, followed by G repeated from 0 to 350,000 times, followed by F; the pattern GAS(TE){,2}F means GAS, followed by TE repeated from 0 to 2 times, followed by F; the pattern GAS(TE){2,2}F means GAS, followed by TE repeated exactly 2 times, followed by F (If the pattern in the parentheses is an OR expression (see below), it cannot be repeated more than 2,000 times).
The pattern <GACCAT can only be found if it occurs at the beginning of the sequence range being searched. Likewise, the pattern GACCAT> would only be found if it occurs at the end of the sequence range.
The Control panel is limited to the motif entry box in which the pattern should be entered, the 'Search' button to launch the search, the 'Find Next' button to go to the next occurence of the motif, and the 'Return' button to leave the search tool.
When a motif is found, the background of the snapshot window will become black, and the motifs will be highlighted in red.
The 'Tree' tool can be divided in two part. The first part consists in the tree building, which is done through the main Ordalie window. Once the tree is computed, its exploitation will be done in a dedicated new window.
The tree is computed using the FastME program using default parameters. Ordalie computes first a distance matrix based on identity percentages calculated over the selected residue range. Although Bayesian based algorithms seem to produce more accurate trees, FastME is a good compromise between speed and accuracy.
The tree can be computed on a subset of sequences and on a given residue range, for example, a region or a domain.
The left part of the Control panel deals with sequence and residue range selection.
The 'Draw' button launches the computation, and draws the resulting tree in a separate and dedicated window. The 'Return' button leaves the Tree tool.
Each newly computed tree will appear in a new and dedicated window, that allows the exploration of the tree characteristics.
Ordalie is able to render two types of trees : dendrograms and radial trees. Some of the following options are specific to one or the other tree representation (see below).
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The upper part of the tree rendering window is the drawing area, and the bottom part the control panel area.
Adding information to the tree representation :
Tags and tree annotation :
Leaf labels :
Buttons :
Traces of the evolution pressure that maintains the structure and function of a protein family can be found while examining the residue conservation along the alignment. Both global and group conservations may help in deciphering functional sites like binding sites, interaction patches, or specialization coupled with intra-groups organization.
Ordalie offers several methods to compute conservation. Within this tool the user can try several methods to compute residue conservation. The results are temporarily kept until they are saved. A saved residue conservation computation becomes then a new feature attached to the current snapshot and can, as any other feature, be used in any tool allowing feature display.
Many methods exist to compute conservation, and they have been tested and compared extensiveley [12,4]. Ordalie implements some of them, as well as two home-made conservation methods.
The available automatic mathods are :
From left to right in the Control panel :
Features in Ordalie may come from the original alignment file (Macsims/XML or ORD files), from within Ordalie (residue conservation computation for example will create a new feature), loaded from the feature file format (see section 6.4) or defined by the user. This tool is dedicated to feature management.
The Control panel of the 'Features Editor' is really simple.
It consists, from left to right, in :
![[*]](/usr/share/latex2html/icons/crossref.png){kind=icon}
.
It is important to understand the difference between a Feature and an Item of a Feature. Here, a Feature represents a set of instances of a given sequence characteristic that may be distributed over the whole snapshot. A Feature Item, or Item for short, is one instance of a Feature for a given sequence at a given place in the snapshot.
Contrary to all other tools, it is possible to interact directly with the features inside the snapshot window. A right click makes a contextual menu pop up, allowing several actions.
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After having selected Items(s) or region, several option are then available.
If the 'Feature Name' entry is filled with an already existing feature, then the new item will be added to the item list of that feature. If the 'Feature Name' does not exists, a new feature is then created. In all cases the user is supposed to give to the item at least a Color and optionally a Score and a Note.
One of the strengths of Ordalie resides in its ability to link/map features to the 3D models (when available) of proteins. To exploit at best the feature mapping it is essential to proceed in the scope of the structural differences observed between proteins. To achieve that, Ordalie allows to superpose the strucure according to feature, and/or user defined residue range.
A protein structure can be made of several chains, which may be identical or not. A chain is usually composed of an amino acid polymer and ligands (in Ordalie, water molecules are considered as ligands). It is important here to understand that, although superposition computation are done using the polymer sequences, the entities that are moved (superposed) in Ordalie are the entire chains.
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The chain superposition is done in three steps :
The detailed superposition algorithm is presented in Appendix 6.5.
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Suppose the loaded alignment concerns a protein known to be a homodimer (an 
structure) under biological conditions, and for which several 3D structures of some proteins coming from different organism have been solved. By investigating PDB ID (say 1abc and 1def), it is also known that all structures are made of two chains, A and B.
When loading the alignment, Ordalie will recognize the two PDB ID through the sequence names PDB_1abc_A and PDB_1def_B and will then download from the PDB web site the two structures with atomic coordinates, and store them inside a dedicated database. Note that Ordalie knows the coordinates for all the atoms of ALL chains of the structure, not only A. Several cases may be encountered when performing a superposition :
Ordalie doesn't know anything about monomers, dimers, multimers in general. It is up to the user to provide the information, by giving Ordalie the sequences of the chains of interest.
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If the alignment contains 'PDB\_1abc\_A', PDB_1abc_B, and PDB_1def_A, PDB_1def_B, it is then possible to superpose the two dimers. A first superposition step where only PDB_1abc_A and PDB_1def_A are selected will bring PDB_1def_A on top of PDB_1abc_A. A second superposition step where only PDB_1abc_B and PDB_1def_B are selected will bring PDB_1def_B on top of PDB_1abc_B.
Ordalie contains a collection of tools that can be called at any time, and that can stay alive whatever happens in the main window.
The 'Snapshot Overview' is one of the available schematic representation of a snapshot. When launched, a window appears with the schematic alignment at the top and a control panel at the bottom. Any number of Overviews can be launched for a given snapshot.
In this frame, the alignment is schematized by replacing every residues by a grey pixel over a white background. It is then possible to map any feature on top of this scheme. Clicking anywhere on the scheme will automatically centre the main snapshot window on the corresponding position. On the scheme, a stippled rectangle encompasses the region shown by the main window.
Below the alignment frame, a control panel allows to interact with the scheme.
The combobox on the left is a feature selector. By default, it is set to 'automatic', meaning that all features drawn or removed in the main window will automatically be drawn or removed in the Overview window. Selecting any feature in the combobox will simply display it on the Overview.
The '+' and '-' buttons will zoom in and out the scheme.
The 'Print' button will output a PNG file of the schematic alignment in its current state. The user is prompted to give an output file name.
The 'Close' button will close the 'Overview' window.
All information attached to a protein that is not a feature can be viewed and/or edited in Ordalie. Depending on the origin of the alignment (fasta/msf/clustal or Macsim/ORD files) some fields may be empty.
When browsing or editing sequence information, a selector will appear at the top of the window to select the protein of interest. If this protein presents some unusual characteristics (unknown amino acids, the sequence corresponds to a fragment, ...) a red warning will appear on the left of the window.
The information is arranged in four frames.
Some information can be edited to set them or to correct them. Editable fields are : 'sequence name', 'accession number', 'Bank Id', 'description', 'Organism', 'Taxa Id', 'life Domain' and 'E.C.'.
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The changes are applied as soon as the 'OK' button is pressed.
The VRP (Vectorial Representation of Protein) tool is a tool that may be used to define protein characteristics in a graphical manner. The protein sequence is here represented as the path of successive amino acids taken as vectors. The vectorial equivalence of each amino acid is given by a multidimentional scaling of the PAM250 similarity matrix [2].
When opened, the top part of the window displays the VRP of the first protein in the snapshot. Each dot corresponds to an amino acid, and clicking on a dot with <Button-1> display its name and position in the sequence. The VRP can be moved around by dragging the mouse with <Button-1> down. Dragging the mouse with <Button-3> down will zoom the drawing in and out.
On top of the Control panel is the sequence of the currently selected protein. If a dot has been picked in the drawing area, its corresponding residue will be displayed with a red background in the sequence window. By clicking on a residue in this sequence window, its corresponding dot will be displayed and labeled in red.
Sequence selection is done through the 'Sequence' combobox. At the top of the combobox there are items named 'All' and, if applicable, 'GroupX' where X is an integer indicating the group number. This allows the display of the VRP of the whole snapshot or of the groups if present. The group VRP is done by drawing, for each column of the snapshot, the average vector of the column scaled by the number of residues inside the column.
By checking the 'Overdraw' checkbox, the display is not cleaned between each VRP rendering, allowing the display of several VRPs at the same time.
The 'Feature' combobox will select a feature to be mapped onto the VRP drawing. No feature is mapped when dealing with a group. The 'Circle' button displays the amino acids vectors used to build the VRP, the 'Print' button creates a PNG image of the current VRP drawing and the 'Close' button closes the window.
The 3D Viewer is one of the most useful tool in Ordalie. Although it does not offer all the features and functionalities that would a proper Molecular Visualization program like VMD or PyMol, it can be of great help in understanding protein features in the framework of protein structures.
The Ordalie 3D Viewer is organized around the 'Molecule' and 'Object' notions. A 'Molecule' consists in all the chains (and consequently residues and atoms) that are present in a given PDB entry. An 'Object' belongs to a Molecule, and can be a composition of several elements (full chains, parts of chain, residues, ligands, etc ...) belonging to that molecule. Objects can be painted with several colours and can contain several kinds of representation. Feature mapping only applies to objects.
By default, Ordalie will create 3 objects per molecule :
Ordalie is able to represent a structure in several ways.
The 3D Viewer window can be divided in 4 parts. The top of the window is used to display information about picked atoms. Below is the 'Quick Mapping' panel. Below this panel, from left to right are the 'Molecular Objects' frame, the main 3D window, and the 'Actions' panel at the right. The 3D window can be maximized by hittin the <F1> on the keyboard, and hitting <F1> again gives the window its original geometry. All panels may be switched on or off by hitting the <F2> key.
The four comboboxes of this panel allow to make a quick mapping of features on a given molecular object. The left outmost combobox selects the molecule, then the object onto which the feature will be mapped. There are then two features selectors. It is possible to map two features on a same object by selecting one feature with 'Feature 1' combobox, and a second feature with the 'Feature 2' combobox. The features are drawn in order, feature 1 before feature 2. Care should be taken when selecting Feature 1 and Feature 2 as Feature 2 can completly cover Feature 1. For example, if Feature 1 is set to conservation, which implies residues colouring, and Feature 2 is set to PFAM-A , a lot of conservation won't be seen as a PFAM domain extends to a large range of residues. In this case, Feature 1 should be set to PFAM, and Feature 2 to conservation.
Below the 'All On' and 'All Off' buttons that switch on and off all objects that have been defined in all Molecules is the list of all 3D molecules present in the alignment. Aside the molecule name is the 'New' button that allows the definition of new objects for that molecule (see section 'Object Editor' 4.4.4 below). Clicking on a molecule name will open/close the list of the objects defined for that molecule. An object coloured in green is switched on and is displayed on the screen, a red object name means the object is switch off. Each object name is followed by the 'Edit' and 'Del' buttons, used to redefine and delete the object respectively.
This window contains the 3D objects themselves. The objects can be manipulated by the mouse through an arcball system, that is a virtual trackball. All the objects of the scene are enclosed in a sphere, and the objects are moved by dragging the sphere up and down and left to right, the mouse mimicking the hand that would roll the sphere. The mouse wheel is used to zoom in and out the scene. A right drag with <Button-3> will translate the scene in the x-y plane. A <Control-B1> click will show the label of the atom being below the mouse pointer.
Although the Ordalie 3D Viewer tool is not intended to be a complete Molecular graphics program, it still offers some functionalities which are, from top to bottom :
An object is an ensemble of residues and/or ligands belonging to one or several chains, and displayed in given styles with given colours. The Object Editor can be invoked to create a new object (the 'New' button) or to edit an existing object (the 'Edit' button).
In case of a new object creation, the new object name should be entered in the top entry box. Two objects can not have the same name.
The object edition can then be done in a five step process :
Finally, it is possible to add the molecular surface surrounding the object atoms.
When applying a color or a representation style, the user should specify the residues it should apply to. There are three ways to do so :
The object is then finished by clicking on the 'OK' button. The new object will be added to the object list on the corresponding molecule.
This representation can render several selected sequences and features on the same page. The sequences are not schematized as in the Snapshot Overview representation, but are shown as they appear in the alignment window.
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The top of the 'Feature Summary' window is made of a listbox containing the sequence names on the left, and a drawing area on the right. Clicking on a name selects or unselects the corresponding sequence. Multiple sequences can be selected by holding the <Control> key down while clicking on their names with the mouse.
In the drawing area, for each sequence the sequence ID is written on the left, followed by the position of the first residue in the current sequence line, the amino acid sequence itself as present in the alignment, and the position of the last residue in the line. When a feature is selected, each sequence line is followed by a feature line, with the feature name beneath the sequence ID, and rectangles below sequence positions where the feature is present.
The Feature Summary can be moved around by dragging the mouse while holding <Button-1>.
Below the Drawing Area is the Control panel. On the left is a spinbox that selects the type of name the sequence should be referenced with, i.e. its sequence name, its accession number or its bank ID, when available. This choice applies in both the listbox and the drawing area. Follows the font size selector, and then the 'Features' selector. Any number of features can be selected by checking the button corresponding to the desired feature. The 'Notes as Balloon' checkbutton renders or not the note attached to each feature as a flying balloon when the mouse pointer is over the feature. The 'Print' button will ask for a file name that will contain a PNG image of the current drawing area, and the window will disappear by clicking the 'Close' window.
The Barcode alignment tool is an other schematic representation of the alignment.
This chapter describes the Ordalie menus. Only the entries which have not been already explained in this manual will be described in details here.
Opens an alignment file. Several file formats are recognized :
A first format checking is done using the file extension (.msf, .tfa/.fasta, .xml, .rsf, .aln or .ord recognized). Then Ordalie will check the file content. In case of incompatibility, the user will be asked for the correct file format.
Save the entire current alignment in the current file format with the current file name. The first time 'Save' is invoked, the user will be prompted to enter a file name.
Ask for a file format and a file name to save the entire current snapshot.
Save only the visible part of the snapshot in the specified file format and file name.
Close all snapshots and all dependencies (Overview, 3D Viewer ...), and set Ordalie ready to load a new file.
When the 'Print' option is invoked, a 'Setup' properties window will pop up. Several printing parameters can be set :
Guess ...
Cuts the selected sequences.
Copies the selected sequences into the memory buffer.
Pastes the sequences present in the memory buffer. The sequences are pasted just below the current selected sequence. If no sequence is selected, then no sequences are pasted. Note that in case of pasting sequences that already exist in the snapshot, their names will be suffixed by '__<n>' where '<n>' is the copy number.
Access the 'Preferences' panel. This gives acces to Ordalie internals.
Increases the font size. The Ordalie default font type and size can be defined through 'Edit' -> 'Preferences' -> 'General'.
Decreases the font size
Most of Ordalie information and computations are logged. The Log window allows to access the crude log text and to save it as a raw text file.
This will output the whole Ordalie log. A cascade menu allows to select between a HTML or Text format. The output file name is the rootname of the original alignment file suffixed by the format type.
Toggles the main window between its actual size and full screen size. This can also be achieved by hitting the <F1> key on the keyboard.
Displays or hides the Icon bar buttons frame. This is useful when a maximal window size is required, for alignment editing for example.
This turns on and off the scores frame. When displaying a conservation score, either through the Features buttons or inside the 'Conservation' tool, the global score and groups scores for each column is plotted.
Swiches on and off the Features frame. This is useful when a maximal window size is required, for alignment editing for example.
In Ordalie, there are three ways of naming a sequence :
In Macsims/Xml and Ordalie file formats, the three types of names are clearly identified. For all other formats, there is only one ID per sequence, and this one will be taken as the sequence name, accession number and bank ID.
This option allows to change the displayed name of the sequences to any of these three possibilities if available. By default, the sequence name is displayed.
Launches the Identity tool.
Launches the Search motif tool.
This option is intended to retrieve information from sequence databases. For each sequence, Ordalie will try to connect to its original database (UniProt, NCBI) and fetch information. The retrieved information will appear in the sequence contextual menu or through the Browse information menu item.
Enters the sequence information browser tool.
Enters the sequence information edit tool.
Enters the Sequence VRP tool.
If the 'Life Domain' information is available, then the sequence names will be colored accordingly. By default, Eukaryots are colored in red, Prokaryots in yellow, Archaea in blue, Viruses in black. This color scheme can be changed through the 'Preferences' panel.
For all PDB sequences present in the main window, show or hide the secondary structure information if it exists. The Helices, Strands and Turns information is extracted from the PDB file itself, and the structures are represented in red, green and cyan respectively.
Launches the Editor tool. The Editor is described in details in section 3.1..
Creates an instance of the Overview Window for the current snapshot. See ``Snapshot Overview'' section 4.1 for details.
Launches Conservation computation tool.
Launches the Tree tool.
Launches the 'Features Summary' tool. Sequences selected in the main window will automatically be selected in the 'Features Summary' tool.
Launches the feature editor.
Launches the snapshot annotation tool.
Creates a barcode representation of the current snapshot.
Launches the Clustering tool.
Adds a blank line separator just below the selected sequence. Be aware that only one sequence should be selected. Adding separator allow the user to create his own clusters.
Removes the separator just below the selected sequence. Be aware that only one sequence should be selected.
Removes all separators, and unselects all sequences.
Colours residues with the same mapping as in the 'Editor'.
Enters the 'Superposition' tool if several PDB structures are available in the alignment.
Launches the '3D Viewer' tool.
This tool allows coloring the secondary structures according to their sequence identity level in the snapshot. When invoked, a parameter window will pop up with the following parameters to select:
As the 3D structures may be changed using the 'Superposition' tool, this option allows to save a given 3D structure present in the alignment. It may be useful to output the superposed structures in order to render them in a more sophisticated drawing program.
When invoked, a window will pop up to ask the user for some parameters :
This menu item pops up a window giving some information about Ordalie, such as the currently used version, and the URL for the Ordalie home page.
Launches this manual on the default web browser.
| Option | Values | Description |
|---|---|---|
| -convert | <tfa|msf|xml|ord> | Converts the alignment into the format indicated by -convert. The converted output file name will have the form <alignemnt file>.<format> |
| -precompute | <0|1> | :precompute clustering and conservation for each PFAM domain |
| -threshold | <x> | Set conservation threshold level. <x> should be set between 51 and 100 |
| -batch | <0|1> | Run Ordalie without windows and exit when finished |
The core of Ordalie is build around a in-memory SQLite database [3] which scheme is given in figure XXX. Ordalie takes advantage of this underlaying database to store snapshots of alignments and their associated features. The "ordalie" table contains settings parameters saved at exit allowing the user to find the same state when launching Ordalie again. The "seqinfo" table contains sequence information that are not linked to aminoacids positions (length, molecular weight, isoelectric point, ...) The "seqfeat" table is used to store features data mapped onto the residue sequence. Upon loading of a new alignment file, Ordalie creates a first snapshot as being a read-only copy of this alignment stored as the "original alignment" in the snapshot table. This table contains all snapshots created so far along with their name and description. The "seqali" table records the amino acid sequences as they appear in the snapshots. A link table "ln_snapshot_seqali" binds a given set of sequences to a given snapshot. Accordingly, the "featali" table stores features attached to aligned sequences in a given snapshot. A link table "ln_seqali_featali" couple this two tables. The "clustering" and "cluster" tables define a given clustering attached to a snapshot with its name, the method and residue eones used to compute it, and the resulting clusters with their names respectively. The set of sequences defining a given cluster is available through the "ln_seqali_clluster" link table. The "colmeasure" and "colscore" tables correspond to conservation computations (column measurements) with their name and used method, and the conservation groups with name, value for each column of the group respectively. The conservation score for a given cluster is available through the link table "ln_cluster_colscore". Finally, the "annotation" table contains all information relative to annotation the user adds to a given snapshot. The Ordalie (.ord file extension) consists in a database dump.
This method is based on a vectorial representation of the 20 amino acids. This representation can be the same as the one used in the VRP represetnation, or can be for example, a volume/polarity couple. The score for a given column k can be computed then by :
S(k) = nc/nt * |sum_i=1^nV|/sum_1=1^n|Vi|
where nc is the number of residues in the column, nt is the total number of sequences.
This function is bounded by 0. and N, where N is the number of sequences in the alignment.
It is possible to import features into Ordalie through a features file. It is so posssible to add items to an existing feature, or to create a completly new ones.
The feature file format looks like :
# This is an example of a feature file format # # Declare the feature FEATURE MyFeat ?PROPAGATE? ?all|group? # A line starting by \# is a comment line that can be inserted everywhere # # Structure of the feature item : # seq. name;coord. system; start; stop; color; score , note Q65P3D;LOCAL;23;57;red;0.0;first item Q65P3D;GLOBAL;212;345;blue;0.0;second one FLK14Q;local;123;234;red;0.0;one more # Then go to an other feature FEATURE STRUCT P12345;global;2112;2541;green;0.0;add one
To add some items to an existing feature, the feature name should be exactly identical to the one already present in the alignment as feature names are case-sensitive.
Given two sets of atomsic coordinates A and B, the following algorithm will try to minimize the rms distance between A and B by moving B onto A. The algorithm can be separated in the following steps :
The output of this algorithm provides along with the RMS, the orientation matrix, translation vector, and rotations between the two molecules in different forms.
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The translation was initiated by moumou on 2018-08-22
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