Help

  1. General information
  2. How to use RNApdbee
    1. 3D scenario
    2. 2D scenario
  3. RNApdbee routines
    1. Base-pair identification
    2. Handling of missing residues
    3. Handling of non-canonical interactions
    4. Secondary structure drawing
  4. Supported files formats
  5. System requirements
  6. Citing RNApdbee


1. General information
In the RNA structural biology and bioinformatics an access to correct RNA secondary structure representation is of crucial importance. This is true for both, secondary and three-dimensional RNA structure prediction studies. RNApdbee is aimed to derive secondary structure topology from the tertiary structure of RNA and/or from the list of base-pairs. The tool supports processing of large unknotted structures as well as RNAs with pseudoknots. In particular, it allows for the following operations:
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2. How to use RNApdbee
There are two major scenarios of using RNApdbee application:
  1. 3D→(....), in which the secondary structure is extracted from a pdb file,
  2. 2D→(....), where the secondary structure topology is derived from a list of base-pairs.
Both scenarios are based on the algorithm that iteratively unknots the RNA structure, saves partial information about knotting order, to finally merge intermediate results and encode the RNA topology. The result is released in dot-bracket, BPSEQ and CT, together with a graphical image. Let us describe the two scenarios in detail.

3D→(....) scenario

Step 1
In the first step of this scenario, a user should upload the pdb file with RNA tertiary structure. The file can be uploaded either directly from a local disc (use "Browse" button to browse through the local repositories) or from Protein Data Bank. In the second case a user should enter PDB identifier into the edit box, and click the "Get" button. The associated pdb file is automatically downloaded from PDB and made ready for processing by the application.
There are 4 example pdb files stored in the system and ready for processing. They enable new users an easy start with RNApdbee. Uploaded data can be viewed in the textarea after clicking "Show file contents" button, and edited before further processing.

Step 2
In the second step, an application used to identify base-pairs in the uploaded structure should be selected from three available programs: RNAView (default), MC-Annotate, 3DNA/DSSR. 3DNA/DSSR can be selected with additional option "Analyse helices". Additionally, if one wants to have non-canonical base-pairs annotated in the output (either in text and graphical representation, or graphical representation only), an appropriate option should be checked. By default, RNApdbee output representations contain only canonical base-pairs, while non-canonical ones are included in a separate list only.

Step 3
Next, the user should decide whether the graphical image is to be generated at the output, together with the textual representation of the resulting secondary structure. In order to get an image, one should select one of two available procedures: PseudoViewer-based (default) or VARNA-based procedure. Otherwise, 'No image' option must be selected. If the selected procedure fails to generate an image, the alternative one is automatically run.

Step 4
To start secondary structure extraction, the "Run" button should be clicked. This causes an immediate display of the results page with the secondary structure encoded in extended dot-bracket notation, BPSEQ and CT, listing of non-canonical base-pairs with their classification, and a graphical image (if previously requested). The results can be saved to a local disc. Moreover, the same input data can be processed again with the other set of options.

2D→(....) scenario

Step 1
In the first step of this scenario, the user should upload the BPSEQ or CT file from local repository (use "Browse" button to select a file from the required folder). There are also 3 example files in BPSEQ format and 3 example files in CT format available for upload. Uploaded data can be viewed in the textarea after clicking "Show file contents" button, and edited before further processing.

Step 2
In the second step, the user should decide whether the graphical image is to be generated at the output, together with the textual representation of the resulting secondary structure. In order to get an image, one should select one of two available procedures: PseudoViewer- (default) or VARNA-based procedure. Otherwise, 'No image' option must be selected. If the selected procedure fails to generate an image, the alternative one is automatically run.

Step 3
To start secondary structure processing, the "Run" button should be clicked. This causes an immediate display of the results page with the secondary structure encoded in extended dot-bracket notation, BPSEQ and CT, and a graphical image (if requested). The results can be saved to a local disc. Moreover, the same input data can be processed again with the other set of options.
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3. RNApdbee routines
RNApdbee integrates selected functionality of different tools for analysing and visualising RNA structures into a simple and coherent interface. Below, a short note on each of these tools is provided with respect to their roles in RNApdbee workflow.

Base-pair identification
RNApdbee webserver offers the choice between three major base-pair annotation methods, RNAView, MC-Annotate and 3DNA/DSSR. All of them process PDB-formatted data describing the three-dimensional structure of RNAs.

RNAView

RNAView identifies and classifies the types of base pairs and basic RNA motifs such as loops and bulges that are formed in nucleic-acid structures. It provides the implementation of edge-to-edge hydrogen bonding interactions according to Leontis/Westhof nomenclature . The program allows also for identification of tertiary interactions and visualization of 2D diagrams of RNA secondary topology in Postscript, VRML or RNAML format. RNAView can be downloaded from http://ndbserver.rutgers.edu/ndbmodule/services/download/rnaview.html and used after successful installation.


MC-Annotate

MC-Annotate provides a structural graph which encodes geometric information based on atomic coordinates and torsion angles. In general, the structural graph represents the description of every nucleotide conformation (based on sugar puckering modes and nitrogen base orientations around the glycosyl bond), base-base interactions (based on stacking and hydrogen bonding information) and pseudoknots identified in the input structure. MC-Annotate allows also for RNA motif recognition. It is available as a webserver at http://www-lbit.iro.umontreal.ca/mcannotate-simple/ .


3DNA/DSSR

3DNA/DSSR (DSSR: Defining the Secondary Structures of RNA) tool allows to characterize the geometric features of RNAs. It is provided by 3DNA, command-line driven package, which was designed in order to analyse, reconstruct and visualise the three-dimensional nucleic acid structures. 3DNA/DSSR identifies base-pairs of the input RNA structure taking into account modified and unmodified residues that form canonical (Watson-Crick) base-pairs, non-canonical base-pairs with at least one H-bond and non-pairing interactions (e.g. base stacking). Moreover, 3DNA/DSSR characterizes base-pairs using both Leontis/Westhof and Saenger classifications. It detects triplets, higher-order base associations and pseudoknots. Additionally, 3DNA/DSSR provides RNA secondary structure in the dot-bracket notation. It is available as a web server at http://web.x3dna.org/dssr .

Handling of missing residues
The PDB format supports explicit description of missing residues. These are known nucleotides (A, C, G or U) for which however the atom positions are absent. For PDB entries containing missing residues, RNApdbee is able to read a full sequence, but lacks the possibility of deriving the secondary structure. A special character, i.e. the minus sign "–", was chosen to describe missing or unidentified residues in the dot-bracket output for such cases.
Handling of non-canonical interactions
By default, RNApdbee output representations contain only canonical base-pairs, while non-canonical ones are included in a separate list only. Upon user request, non-canonical base-pairs can be also included in text and graphical representations of the output secondary structure. In general, RNApdbee provides information about strong non-canonical interactions (mediated by at least 2 hydrogen bonds between bases). They are annotated in all text and graphical representations. Due to limitations of text formats not all multiplet-involved interactions can be encoded in dot-bracket, CT and BPSEQ. However, they are present in the graphical image. In dot-bracket representation non-canonical base-pairs are printed in bold, while in CT format they are supplied with comments. Additional functions drive classification of non-canonical base-pairs according to Leontis-Westhof and Saenger nomenclatures. An assignment of non-canonical interactions to classes is provided in CT file and in the supplementary table. This table contains also information about weak non-canonical interactions (annotated as "base - base (1H)", "stacking", "base - sugar", "base - phosphate", "sugar - sugar", "sugar - phosphate", "phopshate - phosphate" or "other" in the 2nd column).
Secondary structure drawing
In order to draw secondary structure topology, RNApdbee webserver integrates the functionality of PseudoViewer and VARNA, and supplements it with own scripts that annotate the orders of pseudoknot interactions, and non-canonical base-pairs.

PseudoViewer

PseudoViewer allows for effective visualisation of large RNAs, also these including pseudoknots, as planar drawings. As an input, it accepts the sequence and the secondary structure data in the dot-bracket or paired format. At the output, an url of the generated image file is returned. The graphical image can be prepared in EPS, SVG, PNG, or GIF. PseudoViewer is available at http://pseudoviewer.inha.ac.kr/ .


VARNA (Visualization Applet for RNA)

VARNA is mainly an interactive software for drawing and editing RNA secondary structures. It supports several input file formats, including BPSEQ, CT and others. The main advantage of VARNA is its support for non-canonical base-pairs (Leontis/Westhof nomenclature) and pseudoknots (first extracted maximal planar subset of canonical base-pairs is a scaffold for the rest of the base-pairings). The output visualisation can be extracted in vector and bitmap picture formats including EPS, SVG, XFIG, JPG, or PNG. VARNA is available as the lightweight applet and swing component at http://varna.lri.fr/ .


Pseudoknot annotation scripts

Graphical images of RNA secondary structure generated by PseudoViewer and VARNA are post-processed by the RNApdbee scripts. Their aim is to annotate the orders of pseudoknot interactions in a graphical way. Different colours have been assigned to particular pseudoknot orders:

Pseudoknot order 1st 2nd 3rd 4th 5th 6th 7th 8th
Text annotation (brackets & letter) [] {} <> Aa Bb Cc Dd Ee
Graphical annotation (colours)

Scripts to annotate non-canonical interactions

Additional scripts post-process the PseudoViewer and VARNA images to display non-canonical interactions. Base-pairs that can be represented in text notation are shown either as gray-filled circles (for regular bps) or coloured dashed lines (for pseudoknots, see table of colours above). Gray dashed lines are used to connect multiplet-involved residues and other pairs unrepresentable in text format.
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4. Supported file formats
RNApdbee can process input data from files in pdb, BPSEQ and CT formats. The output data are released in BPSEQ, CT, extended dot-bracket and graphical formats.

  1. PDB file stores various data concerning the three-dimensional structure of a molecule, the experiment for structure determination, authors, etc. The detailed description of this format is given here . In RNApdbee , only the information about tertiary structure of RNA is considered.

  2. BPSEQ file contains information about base pairs, stored in three columns: 1st column contains the sequence position (starting at one), 2nd column contains the base encoded in one-letter notation, 3rd column contains the pairing base (if base from 2nd column is paired) or zero (if base from 2nd column is unpaired).

  3. CT (connect) file is column based and contains the information about base pairs: 1st column specifies the sequence index (starting at one), 2nd column contains the base in one-letter notation, 3rd and 4th columns specify additional indices (the index of preceder and follower of base in the chain; if one of them is zero, it represents the terminal base in the chain), 5th column gives the pairing base (if base from 2nd column is paired) or zero (if base from 2nd column is unpaired) and 6th column corresponds to base number. Additionally, if 7th column appears and starts with a '#', the rest of the line contains a comment.

  4. Dot-bracket notation is used to encode RNA secondary structure topology. Standard dot-bracket encodes nested RNAs only. It is a string composed of dots and brackets, where an unpaired nucleotide is represented as a dot ".", and a base pair is represented as a pair of opening (begin) and closing (end) brackets, i.e. "(" and ")".
    The extended dot-bracket is applied to represent knotted secondary structures: squared "[" and "]" brackets are used for lower-order structures, the curly brackets "{" and "}", angle brackets "<" and ">" and consecutive alphabet letters "A" and "a", "B" and "b", etc. represent higher orders and most complicated pseudoknots. Additionally, the minus sign "–" is used to encode an unidentified residue.

Example encoding of RNA secondary structure:

  BPSEQ format     CT format     dot-bracket notation     graphical image
  1   G   8  
  2   G   7  
  3   C   0  
  4   A   0  
  5   U   0  
  6   U   0  
  7   C   2   
  8   C   1
  8
  1   G   0   2   8   1  
  2   G   1   3   7   2  
  3   C   2   4   0   3  
  4   A   3   5   0   4  
  5   U   4   6   0   5  
  6   U   5   7   0   6  
  7   C   6   8   2   7  
 GGCAUUCC
 ((....))
example_formats

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5. System requirements
RNApdbee is designed to work with most of the available web browsers. The latest versions of browsers are strongly recommended:

Operating system Recommended browser
Windows Microsoft Internet Explorer (8.0 and later), Mozilla Firefox (3.6 and later), Opera (10.53 and later) or Google Chrome (5.0 and later)
Linux Mozilla Firefox (3.6 and later), Opera (10.53 and later)
Mac Mozilla Firefox (3.6 and later), Opera (10.53 and later)

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6. Citing RNApdbee
Any published work which has made use of RNApdbee should cite the following paper:
M. Antczak, T. Zok, M. Popenda, P. Lukasiak, R.W. Adamiak, J. Blazewicz, M. Szachniuk. RNApdbee – a webserver to derive secondary structures from pdb files of knotted and unknotted RNAs. Nucleic Acids Research 42(W1), 2014, W368-W372, (doi:10i.1093/nar/gku330)
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