-> ProteinNetVis tutorial and help

 

The 2 minutes introduction - to get a quick feel of the application

 

Installing

Creating a pathway

Drawing the pathway

Augmenting an existing pathway

Loading experimental data

Continuing a saved pathway project

Using an existing pathway structure

Considerations

Opening a pathway

Working with a pathway

Exploring

Selecting

Filtering

Adjusting the visualization


 

The 2 minutes introduction

 

Download the application with examples from [Download]. Unzip it into a folder of your choosing and run ProteinNetVis.exe.

Choose "Open Pathway" and select from the examples folder the file t-cell1.pth. Wait until it loads.

The orange proteins form the signaling pathway model. The yellow ones are experimental proteins. The small black ones are proteins extracted from HPRD.

Zoom with the mouse wheel, grab and drag the pathway with the right mouse-button.

Click on a protein. Information is shown on the left.

Double click a protein in the pathway. The exploration plane pops up. Click on an interaction or protein for more information on the left. Double click one of the neighbor proteins to move to it.

Press ESC or go to Analyze->Protein->"Stop Exploring" to get out of the exploration plane.

Double click an experimental protein in the right tab to go into exploration mode. Close exploration mode.

Browse through the experimental tabs on the right (Experiment 1, Experiment 2, Experiment1/Experiment2).

Go to Analyze->"Filters and Selectors", select the filter "Protein Connectivity" and click "Add filter". Move the slider at two. All proteins connected to a single protein are removed. Click "Rem" next to the filter slider to delete the filter.

Now choose the selector "Protein Annotation" and add it. Drag the slider to "Kinase" to select all kinases and their interactions. In the "Selections" group box below click the "No Edges" checkbox - now only the proteins are selected. Click the "Interactions between" checkbox to select interactions between kinases. While holding the control button down right click anywhere on the canvas - select "Change shape" and choose hexagon from the combo. All the kinases are now shown as hexagons.

Close the application.

 

 

 

 

Installing

Download the archive from [Download] and unzip it into a folder. Run ProteinApp.exe.

 

Creating a pathway

A pathway can be created in two ways: either by drawing a canonical signaling pathway model or by preprocessing and then augmenting an actual signaling pathway image (such as a JPEG, BMP or PNG). See image below.

The HPRD interaction database comes with the software as flat files. The STRING interaction database is not supported by the currently released version.

If you want to create a pathway by drawing a canonical signaling pathway read [here]. If you want to augment an existing signaling pathway read [here].

To learn about the type of experimental data you can load read [here].

 

Drawing the pathway

[watch movie]

Select the Create Tab on the left.

Check the "Standard" radio-button.

If you have experimental data read  [experimental data]. Load as many experimental files as you have.

Click Next.

Input a file name under which the pathway will be saved.

Wait until HPRD is loaded.

Select "Place Protein" and click on the canvas. Type in the name or abbreviation of the protein in the pop-up window and press "Get by name". Then select the appropriate protein from the list and press "Place Protein". If you simply want to add a label to the pathway click "Just some label" after typing the text. Repeat this process until all the proteins in the pathway are on the canvas.

To move proteins to other positions, deselect all buttons on the left, and then drag proteins on the canvas. Repeat this process until all proteins in the pathway are positioned.

Select "Place Interactions". Select pairs of proteins to place interactions between them.

"Delete Protein" and "Delete Interaction" remove elements from the layout.

Saving from time to time by going to the File tab and pressing "Save" will ensure that you don't lose your work. Pathways can be continued at later times.

Once your pathway layout is complete, press "Next" at the bottom of the left pane.

Input 1 in the "Levels" edit box. Read more about this parameter [here].

You can add a filter to be applied to the resulting network. Applying a filter at this stage rather than after the network is built is recommendable since it will speed up the build process. A negative value in the filters boxes means the filter will not be applied.

Click "Finish".

Wait until the pathway is created and loaded.

On the right the "Analyze" tab will be activated and the "Visuals" tool is selected. The proteins on the canvas will move around slightly to adjust their positions; click adjust positioning to stop the layouting process. Read [Changing visual aspects] to learn more about how to to adjust the visualization (positions, sizes, colors and shapes).

 

Augmenting an existing pathway image

[watch movie]

Select the Create Tab on the left.

Check the "Paint on Image" radio-button.

If you have experimental data read  [experimental data]. Load as many experimental files as you have.

Click Next.

Input a file name under which the pathway will be saved.

Select a pathway image that you want to augment.

Wait until HPRD is loaded.

By holding the left mouse button pressed and dragging, draw a stroke around a protein in the image. The application will attempt to find the boundaries of the element you have circled and mark it. If you are not satisfied with the selection, right click the mouse button and try again. The selection depends on the accuracy of the stroke and some parameters that are adjusted with every selection attempt. Specifically, if the marking is much smaller then the selection area then the system will increase the color gradient tolerance so at the next step a larger area will be marked for the same selection; if the marked area is much larger than the system will decrease the gradient tolerance.

You can also select labels and other shapes such as organelles. They should not be however very large elements of the pathway (such as a membrane), or interactions.

Now, draw a stroke over an interaction. The application will try to match the exact interaction in the image. Again, right click and retry in case of bad selections. Interactions are presumed to run from the vicinity of one protein to the vicinity of another. Please avoid drawing interactions that don't have proteins as endpoints.

Saving from time to time by going to the File tab and pressing "Save" will ensure that you don't lose your work. Pathways can be continued at later times.

Please note that the accuracy of this method is dependent on multiple factors such the image quality, background, shapes captured etc. If you are not able to get good selection try reproducing the pathway using [pathway drawing].

Click "Next".

Now you have to assign HPRD identifiers to the selected protein shapes. Type the protein name or abbreviation and click "Get by name". If the shape is not an actual protein select "Just some label". Once you select the appropriate protein from the list, press "Place protein".

Input 1 in the "Levels" edit box. Read more about this parameter [here].

You can add a filter to be applied to the resulting network. Applying a filter at this stage rather than after the network is built is recommendable since it will speed up the build process. A negative value in the filters boxes means the filter will not be applied.

Click "Finish".

Wait until the pathway is created and loaded.

On the right the "Analyze" tab will be activated and the "Visuals" tool is selected. The proteins on the canvas will move around slightly to adjust their positions; click adjust positioning to stop the layouting process. Read [Changing visual aspects] to learn more about how to to adjust the visualization (positions, sizes, colors and shapes).

 

Loading experimental data

You can currently load experimental data as flat files.

The format of each line contained in the file is:

HPRD_identifier[tab]peptide_sequence[tab]site[tab]value1[tab]value2...[tab]valueN

For an example look [here].

In the pathway creation process there will be a button for adding experimental data. You can load multiple experimental files.

By checking the "All experimental proteins" checkbox you ensure that all experimental proteins will be in the pathway regardless of whether they are connected to the known pathway or not. These unconnected proteins will be shown in the lower right corner of the network.

 

Continuing a saved pathway project

[Opening a pathway] that was saved during the build process and is not complete will allow you to continue from where you left of. Read either [drawing the pathway] or [augmenting pathway image] for details.

 

Using an existing pathway structure to create a new pathway

Because creating a pathway structure is time consuming, you can use an already built pathway to import the structure and then create a new pathway with other parameters or experimental data.

Selcte "Create" from the upper left corner.

Check the "From Existing" checkbox.

If you have experimental data read  [experimental data]. Load as many experimental files as you have.

Input a file name under which the pathway will be saved.

Choose the pathway to use as model.

Wait for the model pathway to load.

You can now alter the structure. When you are done, click "Next".

Input 1 in the "Levels" edit box. Read more about this parameter [here].

You can add a filter to be applied to the resulting network. Applying a filter at this stage rather than after the network is built is recommendable since it will speed up the build process. A negative value in the filters boxes means the filter will not be applied.

Click "Finish".

Wait until the pathway is created and loaded.

On the right the "Analyze" tab will be activated and the "Visuals" tool is selected. The proteins on the canvas will move around slightly to adjust their positions; click adjust positioning to stop the layouting process. Read [Changing visual aspects] to learn more about how to to adjust the visualization (positions, sizes, colors and shapes).

 

Considerations

Please note that reliability of pathway image augmentation is dependent on multiple factors such the image quality, background, shapes captured etc. If you are not able to get good selection try reproducing the pathway using [pathway drawing].

Save the pathway as you are working. Position changes are only retained if the pathway is saved. It is recommended that you save during pathway drawing or image processing.

The "Levels" parameter when creating a pathway influences the size of the pathway. The protein network is constructed iteratively. At each step, new proteins that are connected to the current network are brought in, while the initial network consists of the signaling pathway model. "Levels" essentially specifies the numbers of iterations done.

For example Level = 0 means that nothing will be brought in. The protein network will only consist of the user inputed proteins. Level = 1 means that proteins connected to the user specified proteins are brought in the model. Level =2 means that other proteins connected to the previous ones are brought in.

Depending on the size of the protein interaction database you can choose different levels. For HPRD anything more than 2 levels will yield networks to large to work with.

 

Opening a pathway

Select the "File" tab.

Select "Open Pathway". Select a "*.pth" file.

Wait for the pathway to load.

Note: Make sure the image file associated to the *.pth file (called name_img.png for instance) is in the same folder!

 

Working with a pathway

Once a pathway created or loaded, the "Analyze" tab on the right becomes active and you can work with it using the operations below.

Exploring

[watch movie]

Zoom using the scroll wheel of the mouse. Grab and move the pathway by holding the right mouse button down while moving the mouse.

By double-clicking a protein you can enter exploration mode: that protein and its interactions are shown in detail. You can also enter exploration mode by going to Anlayze->Protein->Start Exploring.

You can stop exploration by pressing escape while being focused on the pathway window or by clicking Analyze->Protein->Stop Exploring.

While in exploration mode you cannot click on protein in the lower plane.

Double clicking on a protein in the exploration plane will cause the exploration plane to transition to the other protein.

On the right there is space reserved for the experimental data. Experimentally determined proteins and sites are shown together with heatmaps computed from the experimental data. There are tabs corresponding to each experimental set and tabs for pair-wise experiment comparisons. Double-clicking on one of the proteins in the experimental tab will start exploration for that protein. The size of the experimental area can be adjusted by grabbing and moving a sizer handler located between the experimental region and the pathway display.

By left clicking a protein in any of the viewing plane, you can get more information about it on the left. This works for interactions as well in exploration mode only.

By right clicking a protein or interaction you can change its attributes. If several proteins are selected you can right click while holding control down and change attributes for the entire group.

Multiple selections can be achieved by holding control and clicking on elements or by using [selectors] and [saved selections]. If both interactions and proteins are selected while right-clicking then the main options are for the proteins and a submenu will be available for the interaction properties.

Selectors

[watch movie]

By clicking a protein on the canvas you select it. Hold down control to select multiple proteins. Implicitly, the interactions leaving the selected proteins are also shown.

In the "Analyze" tab, the tool "Filters and Selectors" has a group dedicate to more complex selection options. The "No proteins" checkbox will leave only the edges selected. "No interaction" has the opposite effect. "Interactions between" will limit the interactions shown to those between the selected proteins - this option is only valuable in the case of multiple protein selections.

The "Invert" button will select the proteins that are currently not shown.

The "Expand" button will select the proteins interacting with those currently selected.

You have the ability to save the current selection. Click the "Save selection" button and enter an name; the selection will appear in the selections list. By selecting multiple selections in the list (using control) you can add up selections.

Finally, you can use selectors to perform more complex selecting. For instance, choose the "Protein Connectivity" selector from the combo-box and add it. By dragging the slider you will be able to select proteins that are involved in more or less interactions. To remove the selector click the small "Rem" button next to the slider. "Inv" will invert the selection.

The following types of selectors are currently available:

  • Protein Connectivity: selects proteins that have a number of interactions greater than a threshold.

  • Protein Annotation: selects proteins that belong to a certain class. This filter is based on protein annotation so results may not always be accurate.

  • Path: selects proteins that connect a sub-group of proteins. The sub-group of proteins is specified by a selection that can be the current selection or one of the saved selections. See the image below for a schematic: if the orange proteins are the selection, the left image shows path filter = 1, the right image shows path filter = 2. This is because in the left picture, proteins that are not on paths connecting the selected proteins, or that are on paths longer than one, are removed.

  • Already explored: selects proteins that have been looked at extensively. Every time a protein is visible in the exploration plane, it is considered more explored. The percentage of exploration is also shown in the exploration plane as a small white bar that gradually fills up with red.

  • Pathway proteins: selects pathay proteins.

  • Exploration proteins: selects proteins depending on how many sites are phosphorylated. Selecting one site will thus show all experimental proteins.

Filters

[watch movie]

Filters are based on selectors. They remove proteins from the display based on some criteria. The same filters as selectors are available. While only one selector can be active at a time, filters can be stacked on top of each other. Read [Selectors] for details that are also applicable to filters.

 

Adjusting the visualization

Several aspects of the visualization can be changed by going to "Analyze"->"Visuals".

First, the way proteins are placed can be automatically adjusted by clicking "Adjust positions". To proteins will then start moving. To stop this process deselect "Adjust positions". There are three spin-boxes below. By increasing "Push Apart" the proteins will be placed further away from each other. By increasing "Pull together", proteins that are connected will try to get closer together. "Hubs apart" will cause highly connected proteins to move apart.

Sizes can be changed for various visualization aspects by selecting one from the combo box in the sizes group and  varying the slider below.

To change colors, pick a visualization aspect from the "Color settings" combo and pick a color. Transparency for database extracted interactions or for the exploration plane can be varied from the two sliders below.

Finally, the two checkboxes at the bottom of the tab influence exploration layout. "Explore resemble" means that the protein positions in the exploration plane will try to be close to the their positions in the main plane while preserving minimum distances between them. Otherwise, proteins are just placed circularly around the center protein. "Tilted Explore" means that while in exploration mode both the global plane and the exploration plane are in tilted to better suggest 3D and depth.