Øvelse skrevet af: Morten Nielsen
Earlier in the course you have used the BLAST program to perform fast alignments of DNA and protein sequences. As
shown in todays lecture BLAST will often fail to recognize relationships between proteins with low sequence
similarity. In todays exercise, you shall use the iterative BLAST program (PSI-BLAST) to calculation sequence
profiles and see how such profiles can used to
- Identify relationships between proteins with low sequence similarity
- Identify conserved residues in protein sequences (residues important for the structural
stability or function of the protein)
First part. When BLAST fails
Say you have a sequence Query
and you what to make predictions about its function and structure. As seen earlier in the course, you will
most often use BLAST to do this. However what happens when BLAST fails?
Go to the
Now select protein blast option.
Paste in the query sequence
Set the database to pdb, and press
Now go back to the BLAST web-site.
Select the Position-specific iterated BLAST
(PSI-BLAST) option. Paste in the query sequence
Set the database to nr, and press
- Q1 How many significant hits does BLAST find (E-value < 0.005)?
- Answer: Zero
- Q2 How many significant hits does BLAST find (E-value < 0.005)?
- Answer: 16 (excluding the identical match)
- Q3 How large a fraction of the query sequence does the significant
hits match (excluding the identical matches)?
- Answer: About 50%
- Q4 Do you find any PDB hits among the significant hits (search for pdb in the
hit list or look for the colored S to the right of the E-value))?
- Answer: No
Now run a second BLAST iteration. Press Run PSI-Blast iteration 2.
- Q5 How many significant hits does BLAST find (E-value < 0.005)?
- Answer: More than 100
- Q6 How large a fraction of the query sequence does the significant
hits match (do not include the first hit since this is identical to the query)?
- Answer: About 50%
- Q7 Why does BLAST come
up with more significant hits in the second iteration? Make sure you answer this question and understand what is going on.
- Answer: In the first interation BLAST uses the BLOSUM scoring matrix to align and identify
significant hits. Before running the second iteration, the sequences of the significant hits
are aligned and a sequence profile is estimated. That is at each position the frequency of each
of the 20 amino acids is estimated. Now for the second BLAST iteration, this sequence profile
is used as scoring matrix making the search specific for the query sequence.
- Q8 Do you find any PDB hits among the significant hits (search for pdb in the
hit list or look for the red colored S to the right of the E-value)?
- Answer: No
If you did not find a PDB hit among the significant hits, run a third Blast
- Q9 What is the PDB identifier for the best PDB hit?
- Answer: Yes. The hit pdb|1A0P|A Chain A, Site-Specific Recombinase, Xerd. The PDB identifier is 1A0P chain A
- Q10 What is the sequence simularity between the query and this PDB hit?
- Answer: 16%
- Q11 What is the function of this protein?
- Answer: Site-Specific Recombinase. DNA Recombination.
Identifying conserved residues
You have now (hopefully) identified a structural relationship between the Query sequence and
a protein sequence in the PDB database of protein structures. Say you would like to validate this
relationship. This one could do by mutating (substituting) essential residues in the query sequence
and test if the protein function (or structure) is affected by these mutations.
The protein sequence of the query is large (more than 400 amino acids) and a complete mutation study
including all residues would be extremely costly. Instead one can use PSI-BLAST and sequence profiles
to identify conserved residues that are likely to be essential for the protein structure and/or
Below you find a set of 8 residues from the Query protein sequence. You shall use the PSI-BLAST and Blast2logo
programs to select four of the eight residues for a mutagenesis study (you shall select the four
residues based on sequence conservation only).
- (a): H271
- (b): R287
- (c): E290
- (d): Y334
- (e): F371
- (f): R379
- (g): R400
- (h): Y436
You shall use the Blast2logo server to identify
which residues are conserved in the Query protein sequence. Go to the Blast2logo server and upload
the Query sequence. Select the
Blast database to NR70, and press submit (note it might take some (5-10) minutes before your job is completed).
When the job is completed you should see the logo-plot on the website. If the logo does not display, you can download
the image file (click on the Download logo file) and open it from your desktop.
- Q12 Which of the eight residues listed above are most conserved and hence most likely
to be essential for the protein stability and/or function?
- Answer: R287, E290, R400, and Y436 (Note R287, is refering to amino acids 287 which is an R)
You shall use the Phyre program to validate if the
structural properties of the four most conserved residues from question Q12 indeed could form an active site.
Go to the Phyre web-site and upload
the Query sequence and
press submit. Note it might take some (10-20) minutes before your job is completed. To save you time, I have
run the calculation for you. Yoy can find the output here Phyre output.
Find the PDB hit identified by PSI-BLAST.
- Q13 Does Phyre agree that this hit is significant?
- Answer: Yes. The top scoring hit is 1A0P
Download the highest scoring Phyre model, and open the model file in Pymol.
Show the location of the four essential residues from question Q12 on the structure. Could the residue
form an active site?
- Q14 Could the residues form an active site?
- Answer: Yes they are all in close contact and exposed to the solvent.
Now you have seen the power of sequence profiles in general and the PSI-BLAST program in
particular. Using sequence profiles you have been able to identify a relationship
between protein sequences fare below 30% sequence similarity. Further, you have made qualified
predictions on the protein function and selected a set of essential amino acids suitable for
experimental validation of the structural and functional predictions.