Tutorial 4 Substitution matrices and PSI-BLAST

Transcription

1 Tutorial 4 Substitution matrices and PSI-BLAST 1

2 Agenda Substitution Matrices PAM - Point Accepted Mutations BLOSUM - Blocks Substitution Matrix PSI-BLAST Cool story of the day: Why should we care about cellular fusion in worms? 2

3 Studying distant homologies When we study a new organism/ protein we may find a lot of unknown sequences that we would like to characterize. We might not be able to find any close homologies. In the evolution, three-dimensional structures of proteins may be conserved even after considerable erosion of their sequence similarity

4 Multiple alignment of the new protein families of the HSP70-actin fold O-sialoglycoproteases (OSGP) and related proteins Newly identified proteins with the HSP70-actin fold UDPases and extracellular ATPases Classic HSP70 and sugar kinases Aravind and Koonin, Journal of Molecular Biology,

5 Comparison of the HSP70 structure and a structural model of the O-sialoglycoprotease Aravind and Koonin, Journal of Molecular Biology,

6 Substitution matrices model different evolutionary distances. PSI-BLAST enable to find more distant relations between proteins. 6

7 Amino acids were not born equally Both substitution matrices and PSI-BLAST are designed to model the process by which AAs mutate. 7

8 Substitution Matrix Scoring matrix S of size 20x20 Si,j represents the gain/penalty due to substituting AAj by AAi (i line, j column) Based on likelihood this substitution is found in nature Computed differently in PAM and BLOSUM Each matrix is tailored to a particular evolutionary distance. 8

9 PAM vs. BLOSUM PAM Based on global alignments of closely related proteins. The PAM1 is calculated from comparisons of sequences with no more than 1% divergence. Other PAM matrices are extrapolated from PAM1. BLOSUM Based on local alignments. BLOSUM 62 is calculated from comparisons of sequences with no more than 62% identity in the blocks. All BLOSUM matrices are based on observed alignments. They are not extrapolated from comparisons of closely related proteins. BLOSUM are the substitution matrices in use 9

10 Use Recommendations PAM100 ~ BLOSUM90 PAM120 ~ BLOSUM80 PAM160 ~ BLOSUM60 PAM200 ~ BLOSUM52 PAM250 ~ BLOSUM45 Closely Related Highly Divergent Query length Matrix Gap costs <35 PAM30 9, PAM70 10, BLOSUM80 10,1 >85 BLOSUM62 11,1 10

11 Example Query: an uncharacterized (hypothetical) protein Data Base: nr Blast Program: BLASTP Matrices: PAM30 / PAM250 BLOSUM45 / BLOSUM90 11

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14 PSI-BLAST Position Specific Iterative BLAST Aimed to find more distant proteins than BLAST allows 14

15 PSI-BLAST Steps 1. Search a query against a protein database. 2. Constructs a specialized multiple sequence alignment based on the top results. 3. Creates a position-specific scoring matrix (PSSM). 4. The PSSM is used as a query against the database. 5. PSI-BLAST estimates statistical significance (E values) Repeat steps 3-5 iteratively. Iterations Query Search Protein DB Results PSSM 15

16 PSSM The PSSM captures the conservation pattern in alignment and stores it as a matrix of scores for each position in the alignment. This profile is used in place of the original substitution matrix for a further search of the database to detect sequences that match the conservation pattern specified by the PSSM

17 PSI-BLAST Example Cellular DNA polymerase enzymes tend to dissociate from DNA after adding a few nucleotides and require an accessory factor to tether them to DNA while elongating the growing DNA chain. In eukaryotes: PCNA In prokayotes: β-subunit of DNA polymerase encoded by the dnan gene

18 E.Coli (dimer) Human (trimer) 18

19 Querying the human protein Changed to

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22 Summarize results by organism 22

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26 Marked in yellow are sequences scoring below threshold on previous iteration 26

27 Iteration 2 27

28 Iteration 3 28

29 Iteration X Hydrophobic AAs: V, L, F, A Polar AAs: E, D, K,R, N, Y Alignment of human proliferating cell nuclear antigen (PCNA) and Escherichia coli DNA polymerase III β-subunit. 29

30 Example 2 We will use a sequence of an uncharacterized (hypothetical) protein: 30

31 Threshold for initial BLAST Search (default: 10) Threshold for inclusion in PSI-BLAST iterations (default: 0.005) 31

32 The results are all hypothetical proteins 32

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34 Cool Story of the day Why should we care about cellular fusion in worms?

35 Cellular fusion In cellular fusion two cells unite and form one cell Fertilization Muscle cells are composed of rows of fused cells Placenta is made up of powerful multinucleated cells that are actually numerous individual cells that have fused The eyes' lenses are formed of rows of fused cells In bones too cellular fusion occurs. The fusion processes are also involved in cancer, viral infections and stem cells. 35

36 Cellular fusion in C.elegans How exactly two cells fuse is not is the focus of work in Prof. Podbilewicz's lab. The worm suits cell fusion research because intensive cell-cell fusion processes take place in its skin and can be easily followed. They identified the protein responsible for the worm's fusion activity - the EFF-1 protein. The researchers showed that in mutant worms skin cells do not fuse and the cells begin to migrate through the body. Beni Podbilewicz 36

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38 ...we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells 38