TITLE: idpr Workflow
AUTHORS: William McFadden, Judith Yanowitz

[DESCRIPTION]
This protocol details about idpr workflow.

[GUIDELINES]
References
Paper Citations
 

 

 

 




R / Package Citations

 

Additional Information: 
Session Info
 

Runtime
 


[STEPS]
SECTION: Installing idpr: Downloading the Current Release
1. 
idpr is published in Bioconductor where the stable, released version of the package can be downloaded. The development version, which may be unstable, is published on GitHub.
The package can be installed from Bioconductor with the following line of code. This requires the BiocManager package to be installed.

 

SECTION: Installing idpr: Downloading the Development Version
2. The most recent version of the package can be installed with the following line of code. This requires the devtools package to be installed.

 

SECTION: Installing idpr: Loading idpr
3. Once installed, idpr can be loaded with the ‘library’ function.

 

SECTION: Installing idpr: Loading idpr
3.1. To test the package is loaded, the idpr function ‘netCharge’ will be used to determine the charge of Glutamic Acid (E) at pH 8. Since pH » pKa, the charge of E should be near -1.

 

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5. To get the ‘idprofile’, a simple function is needed with the sequence and Uniprot ID specified. This generates
all plots in figure 1.

 

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.1. 
 
  

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.3. Calculation of Local Charge in alpha−Synuclein.
Window Size = 9 ; Net Charge = −9.048
  

 

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.4. Measurement of Scaled Hydropathy in alpha−Synuclein.
Window Size = 9 ; Average Scaled Hydropathy = 0.455
  

 

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.6. Prediction of Intrinsic Disorder in alpha−Synuclein.
By IUPred2A long
  

 


SECTION: alpha-Synuclein Figures: Generating Supplemental Figures for alpha-Synuclein
6. The following code generates plots for supplemental figure 1.
SECTION: Generating Supplemental Figures for alpha-Synuclein: Charge-Hydropathy plot of protein domains
6.1. To add multiple points to the charge hydropathy plot, first the sequence will be split into the N-term (residues
1-103) and C-term (residues 104-140). To do this, I will use the ‘AAString’ function from Biostrings. For
the split sequences and the full length sequence, the average net charge and the mean scaled hydropathy are
calculated and put into a data frame. These coordinates will be used for adding ggplot2 annotations. Since
idpr depends on both of these packages, they should already be installed.

 
 
      R  H  Name  Name_Expression    0.047803  0.495049  1-103  aSyn [1-103]    -0.37783  0.34473  104-140  aSyn [104-140]    -0.06463  0.455321  1-140  aSyn [1-140]  
 
SECTION: Generating Supplemental Figures for alpha-Synuclein: Charge-Hydropathy plot of protein domains
6.2. Then, the ggplot is made and annotations are added. See ggplot2 for annotation options.

 
  

SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7. Several point mutations in the alpha-Synuclein NTD have been identified that are linked to familial parkinsons disease. These are annotated here in the context of intrinsic disorder predictions from IUPred2. Functions from ggplot2 are needed for annotations, and therefore this package must be attached if not already.
SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.1. First, the data is retrieved from IUPred2. Setting plotResults = FALSE returns a data frame for custom
plotting.
 
      Position  AA  IUPred2    1  M  0.206376    2  D  0.239899    3  V  0.30533    4  F  0.328603    5  M  0.281712    6  K  0.239899  
 
SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.2. Then, a sequence map is created with the IUPred results. Column 2 (a_syn_iupred_df$AA) is a character
vector of individual, single-letter amino acids. Column 3 (a_syn_iupred_df$IUPred2) is a numeric vector
of IUPred2 scores.

 
  

SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.3. For adding annotations to a sequence map, you can get the position within the plot using the idpr function
‘sequenceMapCoordinates’. This helps guide or identify the coordinates for ggplot2 annotations.
 
      Position  AA  row  col    1  M  4  1    2  D  4  2    3  V  4  3    4  F  4  4    5  M  4  5    6  K  4  6  
 
SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.4. Additionally, several annotations are added and the plot themes are edited. See code for all annotations.
SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.5. Adding the labels for Familial Mutations and ‘*’ to add above the mutated residues. These positions are
determined by ‘sequenceMapCoordinates’ and values are added to row (y) value to move annotations above
letters. To center residues, the column (x) values were adjusted by 0.5 or 0.35.

 

SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.6. Finally, the annotated sequence map is plotted.

 

SECTION: Generating Supplemental Figures for alpha-Synuclein: Sequence Map showing Familial Mutations
7.7. Sequence Map of IUPred2 Predictions for aSyn
  

SECTION: p53 Figures: Fetching the amino acid sequence
8. First, I will use the UniprotR package to get the p53 amino acid sequence from the UniProtID. For p53,
the ID is P04637.

 

SECTION: p53 Figures: Fetching the amino acid sequence
8.1. Retrieved Sequence:

 

SECTION: p53 Figures: Fetching IUPred2A
9. To retrieve the IUPred2 long and ANCHOR2 scores, the p53 uniprot is used. 

 

SECTION: p53 Figures: Fetching IUPred2A
9.1. Prediction of Intrinsic Disorder in p53.
By IUPred2A long and ANCHOR2
  

SECTION: p53 Figures: Fetching IUPred2 Redox
10. To retrieve the IUPred2 with redox predictions, the p53 uniprot is used.

 

SECTION: p53 Figures: Fetching IUPred2 Redox
10.1. Prediction of Intrinsic Disorder in p53
By IUPred2 long|Based on Environmental Redox State
  

SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11. First, the characteristic to map in the plot must be calculated. Here, the tendency for each residue to favor
ordered/disordered structures is determined

 
 
      Position  AA  Tendency    1  M  Order Promoting    2  E  Disorder Promoting    3  E  Disorder Promoting    4  P  Disorder Promoting    5  Q  Disorder Promoting    6  S  Disorder Promoting  
 
SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11.1. Then, the sequenceMap is made. Since p53 is a long sequence, the nbResidues are increased in the sequenceMap
for easier viewing.

 
  

SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11.2. To get the coordinates for ggplot annotations, the ‘sequenceMapCoordinates’ function can assist. Since the
default has been changed for nbResidues, from 30 to 79, this must change in the coordinates function to
properly calculate the position of each residue within the sequenceMap.

 
 
      Position  AA  row  col    1  M  5  1    2  E  5  2    3  E  5  3    4  P  5  4    5  Q  5  5    6  S  5  6  
 
SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11.3. Additional annotations are made, see the code and the example from Fig. S1B on working with sequenceMap
and annotations.
SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11.4. After annotations and titles have been added, the plot can be generated.

 

SECTION: p53 Figures: Generating the idprofile for p53
12. To get the ‘idprofile’, a simple function is needed with the sequence and Uniprot ID specified. This generates
all plots in supplementary figure 2.


 


SECTION: p53 Figures: Generating the idprofile for p53
12.2. Compositional Profile of p53
 

 
 

SECTION: p53 Figures: Generating the idprofile for p53
12.3. Calculation of Local Charge in p53
Window Size = 9 ; Net Charge = −5.774
  

 

SECTION: p53 Figures: Generating the idprofile for p53
12.4. Measurement of Scaled Hydropathy in p53
Window Size = 9 ; Average Scaled Hydropathy = 0.416
  

 

SECTION: p53 Figures: Generating the idprofile for p53
12.6. Prediction of Intrinsic Disorder in p53
By IUPred2A long
  

 


SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.5. 
FoldIndex Prediction of Intrinsic Disorder in alpha−Synuclein

 
 
 

SECTION: p53 Figures: Generating the idprofile for p53
12.5. FoldIndex Prediction of Intrinsic Disorder in p53
  

 

SECTION: Installing idpr: Downloading the Current Release
1.1. The UniprotR package is used within this workflow to fetch the amino acid sequences for the proteins analyzed. idpr contains multiple ways to read in sequences, including from .fasta files via Biostrings. To avoid distributing additional files, we are utilizing UniprotR to fetch sequences from the UniProt database.To run this workflow please install UniprotR. UniprotR is not a dependency of idpr, though this workflow exemplifies how the packages can be used together 
SECTION: Installing idpr: Downloading the Current Release
1.2. 
 


SECTION: p53 Figures: Generating the idprofile for p53
12.1. 
 
  

SECTION: p53 Figures: Making the sequenceMap of p53’s sequence structural tendency
11.5. Sequence Map of Residue Tendency for p53.  

SECTION: alpha-Synuclein Figures: Fetching the amino acid sequence
4. First, I will use the UniprotR package to get the alpha-synuclein amino acid sequence from the UniProtID.
For alpha-Synuclein, the ID is P37840.

 
Retrieved Sequence:

 

SECTION: alpha-Synuclein Figures: Generating the idprofile for alpha-Synuclein
5.2.