HYDROID

A stage-by-stage tutorial on analyzing HRF data of a protein-DNA complex and comparing it to a PDB structure.(Example 1)

System: S. cerevisiae centromeric nucleosome reconstituted on a well-positioning 601TA DNA sequence, DNA is radioactively labeled on 3’ end. Maxam-Gilbert sequencing reactions products were run side-by-side with the HRF gel lanes to assign HRF peaks to the sites on DNA sequence. Data set is taken from Shaytan et al., NAR (2017).

This is a generic example that outlines (1) HRF data quantification from a gel image with the 3’ labeled DNA, (2) prediction of theoretical profiles from PDB strucutres, (3) data comparison.

Python files implementing every stage are provided in this directory.

The contents of this directory can be conveniently downloaded by the following command once HYDROID is installed:

HYDROID_get_ex1

Document H-SASA_params.md further outlines details of theoretical cleavage profiles calculation and their dependence on various parameters.

Video tutorial outlining the key stages is available here.

Quantification of HRF gel electrophoresis images using HYDROIDexp.py.

A stage-by-stage usage example:

Stage 1: Extract lane profiles from gel images via ImageJ - see instructions in exp_s1_extract_lp.md.
Stage 2: Assign band peak locations - see details in exp_s2_assign_peaks.py and run it:
```
python exp_s2_assign_peaks.py
```
Stage 3: Assign peaks to DNA sequence - see details in exp_s3_call_peaks.py and run it:
```
python exp_s3_call_peaks.py
```
Stage 4: Quantify cleavage frequencies by fitting a model to the data - see details in exp_s4_fit_model.py and run it:
```
python exp_s4_fit_model.py
```
See fitting results.
Stage 5: Plot cleavage frequencies along DNA sequence - see details in exp_s5_plot_cl_freq.py and run it:
```
python exp_s5_plot_cl_freq.py
```
See the resulting plots.

Prediction of cleavage intensities from PDB-structures using HYDROIDpred.py.

A stage-by-stage example:

Stage 1: Prepare PDB file with hydrogen atoms - see instructions in pred_s1_prep_pdb.md.
Stage 2: Estimate theoretical hydroxyl-footprinting cleavage profiles through calculating H-SASA profile - see details in pred_s2_calc_H-SASA.py and run it:
```
python pred_s2_calc_H-SASA.py
```
Stage 3: Plot H-SASA profiles - see details in pred_s3_plot_H-SASA.py and run it:
```
python pred_s3_plot_H-SASA.py
```
See resulting plots here and here. Also simulated gel lane profiles from theoretical profiles may be plotted.

Compare profiles

Experimental vs H-SASA: run comp_plot_exp_vs_pred.py
```
python comp_plot_exp_vs_pred.py
```
to get comparative plots for top and bottom strands.
Top and bottom strands of experimental profiles: run comp_plot_exp.py
```
python comp_plot_exp.py
```
to get compartive plots between top and bottom DNA strands to assess pseudosymmetry of nucleosome with respect to the two-fold dyad symmetry axis.

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