BIO 224 Laboratory
CSU, Sacramento
Dr. Tom Peavy
February 13, 15 & 20, 2007
Assignment 3 (20 pts)
(due Thurs Feb 22nd at 5pm)
1. Perform a blastp search using a highly conserved human protein as a query (integrin receptor
beta 1, NP_391988.1). Use the default parameters, (nr or non-redundant database, search “All
organisms”, and the BLOSUM62 matrix). In the formatting section, chose to view 500
Descriptions, Alignments and Graphic Overviews (scroll down in the format section and change
the drop down numbers).
A) Scroll down to the last page of the BLAST results and copy and paste the database
info into this document (section begins after the sequences are presented).
B) What was the effective length of the query sequence? How many sequences did the
database examine? What was the effective length of the database? What do these pieces
of information mean?
C) How many database hits occurred in this search? How many HSP’s were
successfully gapped? What does this mean?
D) What was the threshold value used in this search? What does this mean?
E) Examining the graphical and alignment displays on the first page of the BLAST
results, what species and protein had the highest score and E-value (careful not to quote a
hit for the same human protein sequence)?
2. Perform a similar blastp search using the human integrin sequence using the nr database
(non-redundant), but this time search only the “Arthropoda”.
A) Answer the following questions:
i) How many hits were there.
ii) What species and protein has the highest score and E-value? (record into table
found in question C)
iii) Were there any conserved domains? (examine the red link for conserved
domain hits within the format section and follow the link). If so, describe the
domain(s) that was found?
B) Using the above Arthropoda BLAST search, at what score and E value do you
suspect that the alignment is not for a homologous protein (meaning a non integrinrelated protein)? Provide your reasoning.
C) Next fill in the table by repeating the search (same query, same nr database, same
limitation to Arthopoda) using the two additional indicated scoring matrices.
total # hits
best score
best E value
first search, BL62
BLOSUM45
BLOSUM80
Was the same protein identified as the most closely related sequence in each of the
searches?
BIO 224 Laboratory
CSU, Sacramento
Dr. Tom Peavy
February 13, 15 & 20, 2007
E) What was the effect of changing the scoring matrix with respect to the total number
or hits, best score, and their E values. Explain why?
(hint: think about the relationship of the scoring matrices in terms of matches-- which
matrices give the highest scores for exact matches and highly conserved substitutions?)
3. Repeat above problem using a human protein that is poorly conserved, lactalbumin
(NP_002280). Restrict the database to RefSeq and search “All organisms” using the default
BLOSUM62 matrix.
A) Answer the following:
i) How many hits are there.
ii) What species and protein has the highest score and E-value? (careful not to
quote a hit for the same human protein sequence)?
iii) Were there any conserved domains? (examine the red link for conserved
domain hits within the format section and follow the link). If so, describe the
domain(s) that was found?
B) At what point can you discern that you are no longer examining the orthologous
protein in your BLAST search results? Explain.
C) Perform a similar blastp search using the human lactalbumin sequence using the nr
database, but this time search only the “Arthropoda”.
Fill in the chart below:
Arthropoda
total # matches
best score
best E value
BLOSUM62
BLOSUM45
BLOSUM80
i) Were the same proteins identified as the best score/E value in all three
searches?
ii) Were the alignment over the whole length for the highest hits or only to
portions of the lactalbumin protein?
iii) What was the effect of changing the scoring matrix with respect to the total
number or hits, best score, and their E values. Explain why?
4. What different search strategies might you use when studying a highly conserved protein
(problem 2) versus a poorly conserved protein (problem 3)? (think about the various matrices
and databases)