Tutorial

How to input or edit pathways into MetNetDB

by: Yingjun Wang, Dept. of GDCB, Iowa State University

Before starting edit a pathway, get following information ready:

Pathway overview with all interaction connected as shown in textbooks or references.
For each enzymatic reactions, find the EC#, corresponding substrates and products, and co-factors or regulators (google search, textbook, reference or related web sites).
For each enzyme or gene product, find their gene (AT number for Arabidopsis). You can get this information from reference or AraCyc.
Locations for an enzymatic reactions or a gene product.
Confidence for each reaction, the function of a gene product, or the locations
References and comments.
Synonyms for an entity (synonyms for metabolites, genes and gene products, reactions etc.)

It is recommended to list all the information in an Excel files. Once you have the entire information ready, you are ready to edit the MetNetDB.

Edit a pathway and its reactions

Pathway: Cell Wall building blocks (Endomembrane System/Golgi Body)

Reactions: UDP-D-xylose-4-epimerase, UDP-D-xylose synthase, UDP-D-glucuronic acid-4-epimerase

First, Login to the MetNetDB. Then follow these steps

A. Edit Pathway:

Go to the pathway window by clicking “pathway” tab.
Input the name of the pathway you want to edit, and click “search pathway”.
If the pathway (or a similar pathway) is already in the database, go to the “pathway preview” window (see pathway preview below) and find if this is the pathway you need. If it is, or it dose have most information you need, then you can just edit this pathway by using the same name (or you can change its name by “modify pathway”). If it is not, then you need generate a new pathway.

Example, in pathway window, put “cell wall” and search. If no similar record in DB, just put the pathway name “Cell Wall Building Blocks: (Endomembrane System/Golgi Body)” in “name” and click “insert pathway”. Now a new pathway named “Cell Wall Building Blocks: (Endomembrane System/Golgi Body)” is generated in the DB.

Hint: you may want to generate several specific pathways first instead of a big general pathway. After generating several related pathways, you can combine them to a big pathway by “create pathway”. Remember to use “insert pathway” to generate a pathway. “Create pathway” is only used to combine pathways.

B. Enzymatic reaction.

Enzymatic reactions are edited in “interaction” window.

Go to the “interaction” window by clicking “interaction” tab.
Choose “enzymatic reaction” in small “type” pull-down window. This is also the default type in the interaction window.
You can always input a reaction manually by typing in a reaction name, EC# etc. But I prefer to start with choosing EC# in “EC” first because there are so many pre-loaded reactions. It is easier than putting everything manually. Hint: when choosing EC#, be aware that some small numbers such as “5” comes after big numbers such as “12” or “24”. Such 1.1.1.5 comes after 1.1.1.23. First digital determines the order. For example, UDP-D-xylose-4-epimerase, EC# is 5.3.1.5.
After an EC# is chosen, the corresponding enzyme name will be automatically shown in “catalyst”. You can change this name manually if the original name associated with EC is not what you want (they could be synonyms). I suggest using same name for “catalyst” and for the “name” of enzymatic reaction itself. Copy the catalyst name into “name” for the interaction name unless you want them different. For example, EC#5,3,1,5 gives a catalyst name “Xylose isomerase” (there is a list under it, you can see it by pull down the button). You may want to paste the name “UDP-D-xylose-4-epimerase”into “catalyst” instead. You will also need to copy this name into “name” for the interaction.
lick “auto” in the lower middle column. A list of preloaded metabolites will be shown in product “from” and substrate “to” window on the right corner. (for some of reactions, there are too many (or wrong) metabolites with it, we still need to fix it). Remove the one you don’t want by clicking it and then click “<=”.
If a metabolite is not shown, you need manually input this metabolite. Input metabolite’s name in “entity” and click “search”, a list will show up under it. (Hint, when searching an entity, you may add filter by adding “exact” “location” etc. These filters allow giving returns with exact match, and save searching time, but it may miss some return you want.) Once you find the right metabolites, you can add it to substrate by clicking “from”, or to product by clicking “to”. You can also make reaction into “reverse direction”, or “clear” by clicking corresponding buttons.
If a metabolite is not shown in the list, which indicates that there is no such entity in DB. Enter it manually. You can either go to “entity” window to insert a new entity into database. Or, with more efficient approach, simply input the name in “entity” and click “from” or “to” to input it in corresponding product or substrate window. This will automatically generate a new entity in DB. Although you may need to edit it later at “entity” window to include all the necessary information.
Make sure to change the location for interaction, metabolites. (this should be done in a more efficient way, but now we have to change them one by one)
Make sure to change “type” if they are not right. Also put “coefficient” number.
Add synonyms in “synonym” if there is any. Add reference in “reference” window. Change “confidence”.
If this reaction is “reversible”? The default is “Yes”. If no, change it to “NO”.
Finally and most important, choose the pathways to which this reaction belongs in the pathways list (right up corner). Remove the wrong pathways if there is any. If the right pathway is not shown in the list, click “add”, a new pop-up window will show up, choose the pathway from the window by click “add pathways”, this pathway will be loaded into the interaction window.
Once you get everything correct, click “insert interaction” if you want to generate a new interaction. Or click “modify interaction” if you just want to modify an old reaction. Be aware that “modify” will change the reactions in all the pathways if these pathway are on the list in “pathway window.

There is an alternate method editing a reaction, especially if there is an old similar reaction available. Click “search” button next to the “name” on the up left corner. A pop-up window will show up, put the name of the pathway to search, a list of reactions will be shown if there is any. Click the right reaction; The reaction will be automatically loaded into the interaction editing window. Make corresponding change and save it by “insert” or “modify”.

Once you finish editing a reaction, you can visualize it in “pathway preview” (see pathway preview) to make sure if it is correct. You can always back to interaction window to change it.

Following the preview reaction, you can continue to edit the next reaction in the pathway. MetNetDB program will connect reactions in the same pathway automatically. (Hint: some products of a reaction are the substrates for its following reactions, make sure they are the same name and in the same location, otherwise they will not be regarded as the same entity and will not be connected). Again, you can check the connection in pathway preview.

C. Pathway preview.

Open “pathway preview” window by click its “tab”. After the window shows, right click to open a pop-up menu. Click “pathway selector”, then either choose a pathway from the list or “search” it, then click “add pathways” to open the pathway.

In the pathway preview, you can always right click a node to edit it. The node could be an entity, or a reaction. Once you right-click, a menu will pop-up, you can choose the different functions from it. “Edit the selection” will allow you to edit the node you choose.

D. Composition.

Editing composition will allow you to connect an enzyme or a protein to its corresponding gene(s). This is also edited in the “interaction” window. I prefer to finish all the enzymatic reactions in a pathway first before editing composition. Since this is easy to visualize the pathway in preview, and make sure it is correct. Once adding the composition, it is hard to take a close look of pathways because it appears messy with too much stuff in the pathway preview window.

First choose the interaction types from the “type” pull-down button. Depending on how many isoforms or how many subunits for an enzyme (or a functional component), two different types of composition are available. If a same function is carried out by multiple isoforms, then the composition type is “Composition-OR”, which means multiple genes are associated with the same functional component. If multiple gene products constitute the subunits of a functional component instead of just being isoforms, then their relationship is “Composition-AND”. For an enzyme or a component only associated with a single gene product, we can use either “Or” or “And”. Before editing the composition, you need clear the EC# and catalyst from previous enzyme editing window, otherwise it going to cause trouble. We need to fix it. Also, clear metabolites from pervious “from” and “to” window. This should be cleared automatically too if a composition type is chosen.
After choosing the composition type, put the name for the interaction. This name should be the same as the one for enzymatic reaction unless it is not appropriate. Again, you can click search to see if the composition is already in the DB (same rule applied as in the enzymatic reactions). If not, enter information manually.
Search the corresponding AT # in entity window, choose the polypeptide, and then click “from” to the “composition from” window.
Once adding all the polypeptides AT#, search the enzyme name. This name should be the same as “catalyst” name in the enzymatic reaction (once a new enzymatic reaction inserted, DB will automatically generate a new entity with the same name as the catalyst, and its type is protein complex). Choose the right protein complex and click “to”.
Change the location, confidence, pathway etc. just as in enzymatic reaction editing.
“Insert” or “modify” the interaction and finish the editing.

For example, The gene AT1G30620 is for UDP-D-xylose-4-epimerase, input AT1G30620 in “entity”, then choose AT1g30620 polypeptides, and add it into “from”. Search UDP-D-xylose-4-epimerase in Entity, and put it in “to”. Since in this case, AT1g30620 is the only gene for the enzyme, the type could be “And” or “Or”.