The Alignment Editor is not only an integral part of your Jemboss software, it is also available on its own. If you launch Jemboss from a web service, then for the purposes of this tutorial, you only need download the Jemboss Alignment Editor.

The necessary file for completion of this tutorial is available on our FTP site. Please feel free to download it. If you are using Windows, highlight the file you want and click on it with your right hand mouse button. Select the "Copy to Folder" option to copy the file to your local PC. Linux users need to click on the link for the appropriate file and the download manager will do the rest. Save the files to your Demo folder or the one you specified in the File Management Tutorial.

You will need the file:

• HBB_all.aln

If you have done a multiple sequence alignment using the program emma and you are using a Standalone version, a Show Alignment button will appear on the program form and hitting this will automatically display the alignment in the editor. If you have not used emma then follow the instructions below.

Open the file manager by clicking once on the double arrows at the bottom right of the Jemboss window, and click once on the HBB_all.aln file to highlight it. With your right hand mouse button, click once to open the file management menu. Select Open With using your left hand button and then choose the Jemboss Alignment Editor option.

The alignment will appear in the editor window.

The Jemboss Alignment Editor allows any alignment to be viewed and edited using a variety of different colour displays and consensus calculations. It will read in any alignment in ALN or MSF format.

The sequence names are specified on the left hand side of the display, together with the sequence length. This includes gaps and is the same for each sequence. The default colour display is defined for each residue.

Should you wish to make the window larger in order to see more of the alignment, hover over the end of the Alignment Editor Window with your mouse until a double sided arrow (<->) appears and then drag the window in the appropriate direction to elongate it.

The sequence are slightly misaligned at the beginning where some of the proteins have potentially undergone post translational modification. The Methionine residues of both the Sheep and Cow sequences can be aligned with the Fugu sequence.

Click on the yellow M residue at the beginning of the Sheep sequence with the left hand mouse button and whilst continuinug to hold the button down, drag the residue to the left until it has aligned with the Fugu sequence.

Do the same for the Cow sequence so all Methionines are aligned at the start of the alignment.

The conserved EK residues and surrounding motifs are now out of alignment so re-align them using the same technique but dragging the L residue the opposite way.

This re-aligns an obvious motif within the alignment. Residues can be dragged either left or right to emulate an insertion or a deletion.

Using the View menu of the Editor toolbar, select the Find Pattern option. Type GKK in the sequence field and hit FIND. The pattern will display in the centre of the Editor window between residues 60-70.

Mouse over the sequences to see the exact location of each residue as a tooltip and find lysine residue 63 in the fugu sequence.

There seems to be an insertion here in the Fugu sequence that is not replicated in the other proteins. It interrupts the AHGKK motif in that region.

Drag the A residue in position 62 of the Fugu sequence to the right so it aligns with the Alanine residues of the rest of the sequences. Now click on each sequence name with the exception of the fugu sequence and hit the LOCK button on the bottom left of the Editor window.

Drag the Histidine residue in position 64 of any of these sequences to the right for one position so that the motif once again lines up as the HG motif conserved across all sequences.

Hit the UNLOCK button at the bottom left corner of the Editor window to release the sequences.

The LOCK button allows several sequences to be locked for the purposes of editing the alignment. A sequence is added to a group to be locked as the result of a single mouse click. It does not matter whether these sequences apppear in a consecutive order or are separated. To de-select a sequence, click on it once again and it will become un-highlighted and thus de-selected.

To display the consensus sequence, go to the Calculate menu and select the Consensus option.

The consensus sequence appears and is coloured in the same manner as the aligned sequences. As the HBB gene product is involved in a disease caused by a mutation on a surface protein, this alignment can be viewed using a colour display that will reflect this.

Select the View menu and then the Colour by Property option. From this sub menu, check the box beside Red=Surface, Blue=Buried.

The colour of all sequences will alter to represent surface and buried residues in the display. The consensus can now be set to display only the residues that are conserved across all homologues. The colour preference is recorded together with the matrix used in the status bar at the bottom of the Editor window.

Open the Calculate menu once more and select the Set Consensus Options option. In the options box that appears click the cursor once into the Minimum number of identities for there to be a consensus field and delete the default 0 and replace it with 9 and Calculate Consensus. Close the box.

The consensus sequence will alter to reflect conserved positions only. Gaps are represented in grey.

Should this be the desired final alignment, it can be either be printed straight out in hard copy or to an image file that can be inserted in a separate document. In order to make the display clearer, regions of conservation can be highlighted in other ways. This particular display will not need a consensus sequence so it can be removed.

Click with the right hand mouse button on the actual consensus sequence (i.e. not the sequence name) to display the menu. Select the Delete option and the consensus sequence disappears.

Open the View menu and select the Colour Identical/Matches option. To display the conserved residues only, alter the Threshold for positive matches in the box that appears to 12 and select the colour white from the swatch palette displayed by clicking once on the blue box next to the Positive Match Colour. Hit the Set button. Close the box down.

All the conserved residues are highlighted in red in the display. Scroll to the residues 60-70 to see the lysine insertion in the Fugu sequence.

Open the File menu on the Editor toolbar and select from the Print sub menu the Print Image Files option. Select the desired page set up from the print box that appears and hit OK to open the Save box.

Unlike the save options on the main Jemboss application, the default directory does not alter with any directory preferences you may have set (see the File Management Tutorial) and so you may have to browse to find the appropriate directory to save the image in.

Select the desired directory and enter the name of the file to be saved in the File Name field. On the right hand side of the menu select the image type and maximum residues per line.

The default image selection is jpeg format, but this can be altered to png and they are both offered with a variety of extensions. The default residue number of a line is always the maximum that can be printed on that line. If you are not sure what is best to represent your alignment, use the Print Preview option and test different residue maxima.