The image shows a section of an alignment of Fugu and human P450s. At amino acids 9-11 is the highly conserved EXXR motif seen in the K-helix of all P450s. If you scroll down the page you will see one exception to this motif. T2TP contains EIQC which violates the EXXR conservation. However this is a pseudogene and it is not functional. Far to the right if you scroll over all the way you will find the heme signature region of P450s. The first sequence has FSAGKRICVG. The signature is FXXGXXXCXG. Notice that the F is missing in one sequence. That is probably an error in this sequence. Another sequence editor written for Macs is called Se-Al. This one was written by Andrew Rambaut at the Dept. of Zoology at Oxford with support from The Wellcome Trust. It can be downloaded here.

Now start the SeaView sequence editor. The program has a few example files in it. Under the file menu select open MASE. This gives a list of files. Click on Protein.mas. This is a protein alignment file in MASE format. MASE is mentioned by the authors as having lots of room for long names and other descriptive information such as accession numbers, species names etc. This is not included in formats like Phylip where there are only 10 spaces reserved for a name. The authors really like the MASE format for that reason. However, because we will be using Phylip later, we will use Phylip format in this class.

Scroll down using the left side scroll bar and scroll right using the bottom scroll bar. You can see the alignment in full glory. Notice that the names stay on the left as you scroll to the right, so you can always tell what sequences you are looking at. Across the top on the left and right are position counters. These count the alignment position of the left and right edges of the window. These are not amino acid numbers, since the alignment has gaps in it. To get the amino acid number for any amino acid just click on it. The number will be displayed in the same bar as the counters and it will look like this

Seq:26 pos: 126|113 [CYP21 human]

This tells the sequence number, the position in the alignment and the amino acid number from the first position in the alignment (skips counting the dashes) then the name is given in brackets.

To select a sequence click on the name. To select more than one sequence click and drag. To select non-adjacent sequences just click on each one. A second click will deselect the sequence. If you want to clear all your selections to start over, double click on any name. It is often necessary to move sequences in an alignment to put them next to one another for comparison. To move a sequence in SeaView, select the sequence by clicking on it. Move your cursor to the location where you want it to go and control click (hold down the control key and click). This will move your selected sequence to the new location. If you want to move a block of sequences select the block by dragging and do the same thing.

This is a sequence editor, so how do you edit the sequences. Place your cursor at the point where you want to change the sequence. You can add dashes or delete dashes to slide the sequences. If you want to add or delete gaps to a set of sequences or even all the sequences, select the sequences as above and then add gaps to one of them. Gaps will be added to all the selected sequences at the same time. To add or remove amino acids you must go to the PROPS menu and check allow seq. edition. If you do not do this you cannot change any of the amino acids. Also under the PROPS menu you can allow lower case. I often use lower case to be sequence from a different species that I am using temporarily to fill in a sequence gap. For example, if I have found a Fugu P450 and it is missing the last 20 amino acids, but I have that sequence from medaka (Japanese rice fish), then I can add the medaka sequence in lower case to fill in the missing sequence until I can find it later.

The edit pull down menu allows you to delete a whole sequence by first selecting it and then choosing the delete command from the edit menu. From this same menu you may delete all columns that contain only gaps. This often happens when you remove sequences or trim sequence ends. There is also a consensus option button that builds a consensus sequence from selected sequences. The dot plot option can let you know if your sequence is in correct alignment with another sequence. If the two sequences are out of alignment the diagonal between the two will not fall on the strict diagonal (in red). This indicates one sequence is shifted relative to the other sequence. You can see this by adding a moderate gap into one sequence and then comparing it to its neighbor in a dot plot.

The color scheme can be altered from the PROPS menu under colors. The default for a protein alignment is protein colors, but there is also inverted colors. This makes the backgound colored and the letters are black. This forms vertical bars of solid color when sequences are conserved. There is a way to make your own color scheme with up to 10 colors. You can write a short one line text file called seaview.ini that has to be placed in the seaview folder. This file will say

altcolorgroups = KR,ILMV,AGST,ED,FYW,QNBZ,H,C,P

The color order is fixed and is as follows: red, green, yellow, blue, cyan, magenta, salmon, purple, aquamarine, dark-gray (white is used for dashes). In this example, KR will be red, ILMV will be green, AGST will be yellow, etc. If this file is in the seaview folder when the program is started it will allow you to select alt colors from the PROPS color menu. In this scheme, the positive charges are red, the negative charges are blue, the aromatics are cyan, the aliphatic hydrophobics are green. You can imagine other color schemes that emphasize different properties of the amino acids. If you wanted to color all the charges red and everything else green, use

altcolorgroups = KRED,ILMVAGSTFYWQNBZHCP

Once you have altered the file you can save it and you can print it. Before printing, you have to prepare a printout from the FILE menu. This dialog box requests a name for the printout and it lets you select some options, such as how many lines per page and how many amino acids per block and characters per page. Once the printout is saved you can open it in Word and print it. This program does not let you print out the color alignment. For that type of job you need another program such as PRETTY or BOXSHADE.

Now that we know the basics, lets try to get a sequence alignment into this program to view it. You have just made several CLUSTALW alignments and saved at least one called carrier1 in PHYLIP format. Drag this file from the desktop to the Seaview folder. Try to open carrier1 from the SeaView FILE menu. Select open Phylip and find your file. The open command can be problematic on this program. There are several errors that may appear. If the program does not work close it and reopen it and try again. This sometimes clears it. I know that files that would not open once would open later without any changes to the files. This is frustrating, but we have to deal with it. One method I used that seems to work every time is to close all programs down to the desktop level (Word, Internet Explorer, Seaview) then start Seaview and open the carrier1 file.

If the file will not open in Seaview, open it in Word and check the format. PHYLIP format has a line with the number of sequences and a space followed by the number of positions in the alignment. These numbers have to be correct. The sequences start with 10 spaces for names. There have to be ten spaces even if they are empty. Character 11 begins the sequence even if it is with -----. There may be spaces in the alignment (often at every 10 amino acids) and there may be spaces between sets of sequences (equivalent to pages in the alignment). There must not be any extra spaces at the end of lines and the names only appear once on the first page. Below is an example. If you copied the Phylip format output from CLUSTALW and saved it as text only this worked for me.

6 75
89          LPYNLAELQRQQSPG-LGRPIWLQIAESAYRFTLGSVAGAVGATAVYPIDLVKTR-MQNQ
91          LPFNLAEAQRQKASGDSARPVLLQVAESAYRFGLGSVAGAVGATAVYPIDLVKTR-MQNQ
94          ----------------------------------GGIAGLIGVTCVFPIDLAKTR-LQNQ
95          ----------------------------------GGVAGLVGVTCVFPIDLAKIR-LQN-
132         ------------------------------HMTAGAMAGILEHSVMYPVDSVKTR-MQSL
134         ------------------------------HMVAGAVAGILEHCVMYPIDCVKQTRMQSL

ENMKQTLGVTQK---
ELMKQFFHLN-----
DLMQILLRHLQS---
DSVKVYFRLPRPPPP
DSVKVYFRLPRPPPP