Step 4: Is the three-dimensional structure of human rhodopsin known? - continued

When the BLAST search is done, the result appear in a format like this:

The BLAST result

This output means that 31 hits were found in the PDB. That is, there are 31 protein structures in the PDB for which the protein sequence is similar to that of human rhodopsin (our query). The e-Value is a measure of how good the best hit is. A small number means that the result is reliable (and we can safely say that 2,53·10-195 is indeed small).

Exercise 5:

  1. Click on the line with the BLAST result to reveal all the hits with the relevant details. A screen like the one below appears.

    This is the so-called hitlist, the list of PDB entries that are similar to rhodopsin in terms of sequence. They are sorted by similarity. Number 1 on the list has the most similar sequence (sometimes it is exactly the same), this is PDB entry 1f88.

    The hitlist

    Each line in the hitlist contains the following information:
    • Nr: The rank of the result. Number 1 is the best hit, etc.
    • ID: This is the code by which the the hit is stored in the database. In this case 1f88 is the code for the protein structure in the PDB with a sequence most similar to human rhodopsin.
    • Coverage: The coloured bar shows what section of your query sequence (human rhodopsin) fits on the sequence of the database (in grey). You cannot see the grey bar in the first few hits because the sequences of the query and the hit fully overlap. The grey bar is visible in the lower ranked hits.
    • Description: A short text description of the database entry.
    • BitScore and E-value: Numbers calculated by BLAST to indicate the quality of a hit. Higher is better for the BitScore, lower is better for the E-value. The best hit is always on the top of the list.

  2. We will have a closer look at the first hit, 1f88. Click on the coloured bar and then on the two-coloured bar that appears under Alignment.

    You now see an alignment (a comparison) of two protein sequences. Q (query) is the sequence you entered in BLAST, human rhodopsin in this case. S is the sequence found in the database, in this case the protein sequence for PDB entry 1f88.
    The line between the two sequences only contains the letters that are identical in Q and S. Gaps in the line mean that Q and S are different at that position.

    An alignment, click for a larger version

    You can see that the sequence of human rhodopsin is 93% identical to the sequence in 1f88 (325 out of 348 amino acids are identical). Click on 1f88 to see the complete database entry for 1f88 in the PDB. Note: A 3D representation of the protein in the PDB entry may appear depending on your browser settings.

  3. Read the available information and try to figure out what kind of protein 1f88 is. Answer the questions below:
    • What kind of protein is it? Check the CMPND (compound) lines.
    • From which species is this protein extracted? Check the SOURCE lines for words like ORGANISM or SPECIES.
    • What do you conclude: Is the structure for human rhodopsin known?

In the next section we will look at three-dimensional protein structures.

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