RUBI - Rapid UBIquitination sequence detection


Quick Help and References
Description
RUBI is a new web server to detect regions of proteins which are thought to contain ubiquitinated lysines. RUBI uses an efficient and accurate prediction algorithm based on Bi-directional Recursive Neural networks (BRNN's). BRNN's are a sequence based machine learning algorithm which have being found to be useful for other structural predictions . The method is based solely on the sequence and does not use expensive computations to find multiple sequence alignments.
RUBI also calculates some interesting statistics about all proteins submitted and on each protein individually. For the most detailed description of the methods and accuracy see our paper.

Input (Single mode)
E-Mail address
This is not a requirement. However, if you tick the "E-mail notification of results" box then make sure your email address is typed correctly. If no email is supplied do not close the window (unless bookmarked) while waiting for the results.

Sequence Name
An optional title for your submission. This will appear in the header of the output. We suggest you select one for your own records.

Sequence
All sequences submitted must be in fasta format. Invalid amino acid types, spaces, return characters should be ignored by the server. However, we suggest that you keep the input in the following format: 
>P1
MIETPYYLIDKAKLTRNMERIAHVREKSGAKALLALKCFATWSVFDLMRD
YMDGTTSSSLFEVRLGRERFGKETHAYSVAYGDNEIDEVVSHADKIIFNS
ISQLERFADKAAGIARGLRLNPQVSSSSFDL
>P2
MTFSKELREASRPIIDDIYNDGFIQDLLAGKLSNQAVRQYLRADASYLK
EFTNIYAMLIPKMSSMEDVKFLVEQIEFMLEGEVEAHEVLADFINE
>P3
MENKKMNLLLFSGDYDKALASLIIANAAREMEIEVTIFCAFWGLLLLRD
PEKASQEDKSLYEQAFSSLTPREAEELPLSKMNLGGIGKKMLLEMMKEE
KAPKLSDLLSGARKK
>P4
MKKVFYPLACCCLAAGVFASCGGQKKANAQEEPSKVALSYSKSLKAPET
DSLNLPVDENGYITIFD
where P1, P2, P3 and P4 are the sequence identifiers. It is vital that all identifiers are unique. it is advisable to only use characters A-Z,a-z and 0-9 in the identifiers. For an example multiple fasta example see here and for an example single fasta see here.

Once your sequence(s) are in fasta format there are two input types:

  • Paste: Simple copy the fasta sequences (ala "copy and paste" functions available on all operating systems) and paste into the box provided. A limit of less than 3000 proteins is recommended for pasting. This is due to memory resrictions.
  • Upload: uploading a file with multiple fasta sequences is another possibility. This has no memory restrictions since the data is stored on physical devices rather than memory. In theory any number of proteins can be submitted. For example, the human proteome 39151 proteins can be predicted by uploading the fasta file.



    • Options

    RUBI produces a probability of lysine ubiquitination. A decision on what probability cut-off threshold produces 1% false positives and 5% false positives. Although, our method produces true Bayesian probabilities the data used to learn regions was imbalanced and therefore using 0.5 as the probability decision is not recommended. For this reason internal probability thresholds are defined on each predictors training set. The server hides this information from the user by allowing two types of threshold with respect to the training dataset:

    • 5% False Positive Rate (FPR): This is a strict definition for detection. It is the threshold which produces 5% False positives on the training data. It means that approximately 5% of the lysines in your set will be falsely classified to be ubiquitylated.
    • 1% False Positive Rate (FPR): This is a much stricter definition for detection. It is the threshold which produces 1% False positives on the training data. It means that approximately 1% of the lysines in your set will be falsely classified to be ubiquitylated.


    ESpritz is a fast disorder predictor available here. Disorder is available in 3 styles. For a full description of the ESpritz styles and methods see ESpritz the paper. Rubi allows the user to select one of these 3 styles:
    • Short x-ray: This is based on missing atoms from the Protein Data Bank (PDB) X-ray solved structures. If this option is chosen then the predictors with short disorder options are executed.
    • Longer disprot: The dataset used for this definition contains longer disorder segments compared to x-ray. In particular, disprot a manually curated database which is often based on functional attributes of the disordered region was used for this definition. Disorder residues are defined if the disprot curators considers the residue to be disordered at least once. All other residues are considered ordered. If this option is chosen then the predictors with long disorder options are executed.
    • NMR mobility: NMR flexibility is calculated using the Mobi server optimized to replicate the ordered-disordered NMR definition used in CASP8
    Output (Global stats)
    The first page the user sees is a global statistics and download page.
    Statistics on all proteins
    The global statistics are in the following form: 
    Total amino acids	 : ℕ
Total lysines	 : ℕ
Total ubiquitylated lysines	 : ℕ
Total proteins	 : ℕ
Total % Ubiquitylated	 : ℝ
% of proteins with at least 1 Ubiquitylated lysine	 : ℝ
% of proteins with at least 10 Ubiquitylated lysines	 : ℝ
Mean number of ubiquitylated lysines (standard deviation)	 : ℝ (ℝ)
    Available files for download
    Available for download is a Prediction archive (.tar.gz) archive containing:
    • A directory for each protein containing:
      • The newly annotated fasta sequences (_all.fasta extension). The example below should be self explainatory: 
        >sample.fasta
MSGGGDVVCTGWLRKSPPEKKLRRYAWKKRWFILRSGRMSGDPDVLEYYKNDHSKKPLRIINLNFCEQVDAGLTFNKKELQDSFVFDIKTSERTFYLVAETEED
>sample.fasta_disorder
DDDDDDDDOOOOOOOOODDDOOOOOOOOOOOOOOOOODDDDOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOODDDDDDDOOOOOOOOOOOOOOOOOODDDDDD
>sample.fasta_disorder_confidence_label
33322111889999888111888899999999999881111889999999988888999999999999999881111111889999999999999999111222
>sample.fasta.fasta_ubiquitination_confidence_label
XXXXXXXXXXXXXX9XXXX89XXXXXX89XXXXXXXXXXXXXXXXXXXX3XXXX99XXXXXXXXXXXXXXXXXXXX92XXXXXXXXXX8XXXXXXXXXXXXXXX

        The confidence label depends on the probability of disorder, order or ubiquitinated lysine. For example, a label of "2" on a predicted disordered residue means the probability of that residue being disordered was in the range [0.2,0.3). "X" in the ubiquitination line means the residue is not a lysine.

      • Disorder statistic file (.stats extension). See later for individual statistics.
      • The disorder prediction (.espritz extension). Each line of this file is ordered according to the ordering of amino acids in your fasta sequence. Each line contains 1. Disorder/Order symbol (D/O) and 2. the probability of disorder (e.g. "D 0.462056" means the amino acid is disordered with probability 0.462056).
      • The ubiquitination prediction (.rubi extension). Each line of this file is ordered according to the ordering of amino acids in your fasta sequence. Each line contains 1. Ubiquitination/Ordinary symbol (U/O) and 2. the probability of ubiquitination (e.g. "U 0.462056" means the amino acid is ubiquitinated with probability 0.462056).
    The Histogram of disordered segments graph. An example graph is provided below. The x-axis contains the disorder segment ranked by its length. The y-axis contains the length of the disorder segment.
    Distribution of disordered segments
    Distribution amino acids: Also available to download is the distribution of amino acids in any state and in a disordered state. An example graph is provided below. The x-axis contains the amino acid under investigation. The red bars show the percentage that amino acid occurs. The blue bar shows the percentage of the red bar is disordered.
    Distribution of amino acids in any and disordered states
    Frequency of disorder in all and ubiquitinated lysines: The red bar shows the average probability of all lysines in disordered state. The blue bar shows the average probability of ubiquitinated lysines in disordered state. The p-values were calculated using Wilcox rank sum test.
    Distribution of amino acids in any and disordered states Frequency of structure in all and ubiquitinated lysines: The red bar shows the average probability of all lysines in structured state. The blue bar shows the average probability of ubiquitinated lysines in structured state. The p-values were calculated using Wilcox rank sum test.
    Distribution of amino acids in any and disordered states
    Links to individual protein pages
    Finally, links to the individual proteins are available (see next section). An example list is shown below.
    Distribution of amino acids in any and disordered states
    http://old.protein.bio.unipd.it/espritz/ We feel it is important to rank this list considering it may be a very large list. We allow rankings by %disorder, number of lysines, number of ubiquitinated lysines, length and%ubiquitination. Each page of the list contains 10 proteins where the next 10 can be accessed by clicking the next arrow button.



    Output (individual protein)
    The output page presented for each protein gives a quick overview of the proteins disorder and statistics. In addition, secondary structure and linear motifs are presented for each disorder segment.

    Available files and links
  • Input parameters: A link to a summary of the input parameters the user supplied.
  • Fasta sequence: A link to 4 fasta sequences: (i) amino acid, (ii) secondary structure, (iii) disorder, (iv) disorder confidence label and (v) most importantly the ubiquitination confidence. The confidence label gives a measure of the probability of disorder produced by our machine learning algorithm. For example, a confidence of 3 means the probability of disorder lies between [0.3,0.4).
  • Ubiquitination Prediction (with ubiquitination probability). This is the disorder prediction file (with .rubi extension) described above.
  • Protein disorder statistics: A downloadable statistics file with full protein values.
  • Protein ubiquitination statistics: A downloadable statistics file with full protein values.

    • Ubiquitinated and disordered residue and stats:
    This panel consists of two pieces of information:
    1. Amino acid sequence and the final disorder and ubiquitination decision. Residues are labelled every 10 residues, for example: 
                    110
XXXXXXXXXXXXXXYXXXXXXXXXXXXX
      2. means residue Y is position 110 in the sequence.

    2. The statistics for the individual protein (this is identical to the individual downloadable statistic file above).
    Examples

    Here we present 1 single fasta pasted example, all the sequences from Wagner et al. This section should also serve as a tutorial of how to use RUBI.

    Example 1   -    Human p53. UniProt ID: P04637, 393 residues calculations with short option. Input: input page for human p53.

    Multiple protein example 2   -    Human proteins from Wagner et al [3]. 4273 proteins. Download the human fasta sequences, hit browse on the input page and upload the file. This example will be useful for future reference when processing multiple files.

    References


    • RUBI paper: RUBI: Rapid proteomic-scale prediction of lysine ubiquitination and factors influencing predictor performance. Amino Acids, 46 (4), pp. 853-862  (2014)