The following example aims to display a variety of Jemboss features which can be followed immediately. Should you have little or no previous knowledge of Jemboss then please skip forward and return to this example, should you need to, once you reach the end of the chapter.
This chapter contains many practical activities for you to try. Macintosh users please read "click" for any "left click" directions and "ctrl and click" for any "right click" directions.
This example can be followed using either a remote Jemboss server, or a local installation and assumes that the Jemboss window has already been launched.
The following example aims to illustrate use of the Jemboss interface in the investigation of an enzyme involved in the anabolic pathway of lactose in the gram negative bacterium Escherichia coli.
In order to investigate some of the protein properties, the same sequence will be used in a variety of programs. The sequence list (Section 9.7.3.1, “Sequence Input”) enables the sequence to be selected as default by each program accessed.
Open the Tools menu from the toolbar at the top of the Jemboss window and select the Sequence List. Enter the Tools menu of the resulting window and check the box marked Get sequence lengths automatically. Type uni:bgal_ecoli into the top File/Database Entry field and hit return. The start and end of the sequence is displayed after a short interval. Click with the left hand mouse button on the right hand Default box to select the Default option. Close the Sequence List window.
Each time a new program is selected, the protein sequence for the Escherichia coli beta galactosidase enzyme will be automatically inserted into the input field.
Open the local file manager by using a single left mouse click on the double arrow at the bottom right of the Jemboss window and highlight the top folder in the new pane. Right click to retrieve the menu (Section 9.3.6, “File Manipulation”) and select New Folder. Type Practical into the box provided and click OK.
This is the directory into which all files created in this example will be saved.
Type properties into the Keyword Search (Section 9.9.2, “Application Documentation”) field to the bottom left of the Jemboss window and hit the small GO button next to it. Pepinfo is mentioned as a program which plots amino acid properties. Close the information window and type pepi into the Go To (Section 9.4.5, “Go To Box”) field in the middle of the left hand pane and hit return to display the program form in the central Jemboss panel. Leave everything else as default, scroll down and hit the GO button.
The pepinfo.1.png tab displays a variety of amino acid properties and suggests the protein is composed primarily of smaller aliphatic residues. The pepinfo.2.png tab displays three separate hydrophobicity plots and corroborates information on the first display by suggesting no large regions of hydrophobicity .
Ensure the view is of pepinfo.1.png by selecting that tab with a single mouse click and select the File option on the Saved Results (Section 9.6.8.1, “Saved Results Window”) window. Select the Save to Local File (Section 9.3.5.1, “Saving Analysis Results”) option. Open the Practical folder and type bgal_ecoli_pep1.png into the File Name field and hit Save. Place the mouse into the local file manager (Section 9.3.1, “Local File Management”) to see the pepinfo file. Close the Saved Results window.
Scroll through the alphabetical list (Section 9.4.4, “Alphabetical Program List”) in the middle of the left hand pane to the programs beginning with the letter e and left click on extractfeat. Leave everything as default and hit the GO button. Save in the Practical folder as bgal_ecoli.extractfeat.
The above program extracts information on the features of the sequence directly from the Uniprot entry including secondary structural information and the location of the active sites which may be important for any mutation studies.
Close the Saved Results window.
Select the Favourites option on the Jemboss tool bar and from the resulting list Database Sequence Retrieval to display the EMBOSS programme seqret. Hit the Reset button to remove the default sequence and then type uni:bg*_entc* into the newly cleared sequence input field. Hit the GO button to retrieve any beta galactosidase enzymes from a restricted species set of the gram negative bacteria beginning with entc.
The search retrieves two sequences; one is for a beta-galactosidase 2 enzyme and will be removed.
In the resulting Saved Results window, highlight the entire BGAL2_ENTCL protein sequence (including description) and hit the delete button on the computer keyboard. Select File and Save to Local File. Double click on the Practical folder and then type bgal_entcl.fasta into the File Name field and hit Save. Close the Results window.
Look at the Practical folder in the local file manager to see the new file.
In the Alignment menu (Section 9.4.2, “Program Categories”) on the top left of the Jemboss window, select the submenu MULTIPLE and select the program emma. Alter the input section options to list of files (Section 9.4.7.7, “List of Files”). In the sequence input field directly below the one showing the default sequence, drag and drop (Section 9.4.7.3, “Local Files”) the bgal_entcl.fasta file from the local file manager. Hit the Load Sequence Attributes (Section 9.4.16, “Load Sequence Attributes”) button. Hit the Advanced Options button (to the right of the GO button) and scroll down to reveal further parameters.
De-select the slow pairwise alignment parameter (directly under the additional section header). Alter the Multiple Alignment: Gap opening penalty to 5 and the Gap extension penalty to 2. Hit the GO button.
Batch mode allows the analysis to run in the background so further analyses can be launched simultaneously if necessary. Results, however, may not be immediately obvious so an informative batch submission window will be displayed which you can configure to your taste.
Once the Job manager bar (Section 9.6.3, “Job Manager”) at the bottom of the central pane states Jobs:1 completed, left click once to retrieve the program run results. Highlight the correct emma analysis (there will only be one in this example assuming emma has not been run before) in the Current Sessions Results (Section 9.6.4, “Current Sessions Results”) window and hit the Display button.
This displays results in the EMBOSS default FASTA format which is not the best for identifying similarities in the alignment.
Ensure that the emma.aln tab is displayed and use the File and Save to local file options to save the data to the Practical folder as bgal_align.fasta. Close the Current Sessions Results and the Saved Results windows.
In the local file manager window, highlight the new bgal_align.fasta file. Right click and scroll down the resulting menu to select Open With and then the Jemboss Alignment Editor utility.
Select the View menu at the top of the Editor and then the Find Pattern option. Enter LPEL into the field and hit the Find button to reveal a small area of potential misalignment. Close the Find Pattern window.
Mouse over the residues to locate the E. cloacae leucine (L) residue at position 687 and drag it two positions to the right. Select File and Save As to re-save the altered alignment in the Practical folder. Hit the Save button to confirm.