Procedures for using the NCSA Biology Workbench:
Searches, alignments and trees: (hyperlinks enable you to enter the workbench at different steps)
- Entering the Biology Workbench:
- Launch your graphics browser and go to the following URL: http://glycine.ncsa.uiuc.edu (Note in the left-hand menu that you can utilize a series of tutorials within the workbench.)
- Click on the hyperlink labeled Biology Workbench 3.0.
- If you have already set up an account on the workbench, go to step 4. If, however, this is your first time utilizing the Biology Workbench, then click on the Account Registration hyperlink to set up an account. Fill out the account informatio
n and click Register.
- You are now ready to enter the workbench. Click on the Biology Workbench hyperlink. Enter your user id and password and then click the Register button.
Note that you can click on the demo button sessions to find more suggestions.
- Starting a new session or resuming an old session:
- Before you can utilize the workbench, you need to begin a new session or resume a previous session, just as you need to begin a new file for word processing or to continue a previous file in work processing. In other words, you cannot
use the Protein Tools, DNA Tools, or Alignment Tools until you have resumed an old session or started a new session. So, click the Session Tools button.
- You are now ready to start a new session or resume a previous session. To start a new session, click the Start New Session in the window, and then click the Perform Selected Operation button and then go to step c below.
If, however, you wish to resume a previous session, then click the Resume Session in the window; now click the radio button of the session you wish to continue (e.g., myoglobin1); finally, click the Perform Selected Operation
button and then go to step d below.
- Name the session (= file) you are about to begin. In this case, we are going to name the session enolase since we are going to be conducting a protein search, amino acid alignment and tree construction. Now click Start New Session.
- The page that now comes up looks similar to the one you were previously on, except that your new session (i.e., enolase) is now listed with your previous sessions, if you have any. If the radio button for enolase is not already selected, clic
k it now. You are now ready to begin searching for amino acid sequences. So, click the Protein Tools button near the top of the page.
Overview of remaining operations: Since we do not have an amino acid sequence of enolase for comparison, we must find one. Once we have done that, we will do the following: (1) generate a list of proteins with similar sequences by conducting a searc
h for similar sequences; (2) select sequences we wish to align and then align them; (3) finally, construct a phylogenetic tree and distance matrix based on the sequence alignments.
- Selecting a sequence:
- The Protein Tools button should already be selected; if it isn’t, then select it. In the window, select the SRS Multiple Database Search option. Finally, click the Perform Selected Operations button.
- In the Database window of the SRS Multiple Database Search Query Form, scroll down and select SWISSPROT, which, as the last part of its name suggests, is a protein database. (You can select more that one database to search by ho
lding down on the Command key on the keyboard and selecting the other databases by clicking your mouse.) Before clicking the submit button, you must enter your search term in the query form below the submit button. Type in enolase. Click and hold o
n the button that says All Fields; scroll down and release on Key Work. (Below the query form is a short description of how to conduct a search involving multiple terms.) Now click the Submit button.
- The RESULTS page indicates that we have matched 57 unique records. (The number of unique records may be larger for your search.) In the window containing the list of the 57 records, select swissprot: enoa_human – os homo sapiens (human). (You may have to use the scroll bar in the window until you see this entry.) This entry record contains the amino acid sequence for which we will base the rest of our search.
Note the series of buttons at the bottom of the page that you can click to find out more information about the entry you have selected.
Before we can search for records containing similar sequences, we must import to the workbench the entry we selected above. To do this, click the Import to Workbench button. You are now ready to begin search for proteins with similar amino acid
- Searching for records with similar sequences:
- Click the box in front of the record you wish to search anchor your search.
- After scrolling down in the window, select BLASTP which enables us to compare our protein sequence (ps) to a protein sequence data base (ps db).
- Click the Perform Selected Operation button in order to launch the BLASTP application.
- You should now be in the BLASTP interface page. Note that our Selected Sequence(s) is OS HOMO SAPIENS (HUMAN). Now perform the following steps in order to complete our BLASTP search:
- In the Database Selection window, select SWISSPROT.
- Under the Input Parameters section of the page, click the box to Disable Gapped Alignment.
- Scroll down the BLASTP interface page until you reach the portion of the page labeled Output Parameters. Click on the Number of hits to display pop-up button and change the selection from 500 to 250.
- Now click the Submit button, which will now execute your BLASTP search.
- Selecting records for alignment:
- From the window titled Sequences producing significant alignments, we want to select the sequences that we wish to align to the human sequence. After clicking the first selection, you will need to hold down on the command key (Apple key on th
e Mac) while making additional selections. For the enolase activity, select the following 5 records (human acts as the 6th record):
- ENO_DROME (Drosophila melanogaster)
- ENO_METJA (Methanococcus jannaschii)
- ENO_ECOLI (E. coli)
- ENO_BACSU (Bacillus subtilis)
- Now click the Import to Workbench button. This action will import your data so that you can do alignments.
- Conducting a sequence alignment:
- Click the boxes of all of the records you wish to align—in the case of our enolase alignment, click the boxes of all 6 records.
- In the window, scroll down and click on CLUSTALW – multiple sequence alignment.
- Finally, click on the Perform Selected Operation button. This action launches the interface to the CLUSTAL W Multiple Sequence Alignment application.
- We will not, for this exercise, change any of the parameters on this interface page to CLUSTAL W. So, scroll to the bottom of the page and click the Submit button. CLUSTAL W will now perform an alignment.
- Scroll down to the bottom of the page and click the Import to Workbench button so that you will then be able to launch an application to construct the phylogenetic tree(s).
- Tree Construction:
- You are now ready to use your alignments to construct a phylogenetic tree. If it is not already selected, click the Alignment Tools button.
- Now click the box in from of the CLUSTALW – Protein list. Selecting this box acts to select the entire list of records (i.e., all 6 records, in the case of our enolase exercise).
- In the window, scroll down and select CLUSTAL_W – Phylogenetic Analysis with ClustalW.
- Finally, click the Perform Selected Operations button. This will launch the interface to the tree construction application.
- We will not make any changes to the defaults listed on this page. Simply scroll to the bottom of the page and click the Submit button.
- Two different tree formats are drawn.
- If you wish to obtain a distance matrix, click the Back button on your web browser to go back to the previous web page. Near the top of the page, change the Output format to Distance matrix, and then click the Submit button