tutorial 4 comparing protein sequences

Tutorial 4Comparing Protein Sequences

Intro to Bioinformatics

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Amino acids were not born equally

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Comparing Protein Sequences

Substitution MatricesPAM - Point Accepted MutationsBLOSUM - Blocks Substitution Matrix

Advance comparison toolsPsi-BLASTPhi-BLAST

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Substitution Matrix

Scoring matrix S20x20 for protein alignment (Amino-acid)

Si,j represents the gain/penalty due to substituting AAj by AAi (i – line , j – colomn)Based on likelihood this substitution is found in

natureComputed differently in PAM and BLOSUM

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Computing probability of Mutation (Mi,j)

PAM - Point Accepted MutationsBased on closely related proteins (X% divergence)

Matrices for comparison of divergent proteins computed

BLOSUM - Blocks Substitution MatrixBased on conserved blocks bounded in similarity (at least X% identical)

Matrices for divergent proteins are derived using appropriate X%

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PAM-1

Captures mutation rates between close proteins1% divergenceMi,j = AB / #A

Problematic when comparing far proteinsThe 1% divergence does not capture more sporadic mutations

PAM250 is theoretical (extrapolation based)

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PAM-1

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Captures mutation rates between divergent proteins

Why is BLOSUM62 called BLOSUM62? Basically, this is because all blocks whose members shared at least 62% identity with ANY other member of that block were averaged and represented as 1 sequence.

BLOSUM62

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BLOSUM62

The idea of BLOSUM matrices is to get a better measure of differences between two proteins specifically for more distantly related proteins.

Similar AA have high score

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PAM & BLOSUM

PAM BLOSUMBased on global alignments of closely related proteins.

Based on local alignments.

The PAM1 is calculated from comparisons of sequences with no more than 1% divergence.

BLOSUM 62 is calculated from comparisons of sequences with at least 62% identity in the blocks.

Other PAM matrices are extrapolated from PAM1.

All BLOSUM matrices are based on observed alignments. They are not extrapolated from comparisons of closely related proteins.

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PAM100 ~ BLOSUM90 Closely RelatedPAM120 ~ BLOSUM80PAM160 ~ BLOSUM60 PAM200 ~ BLOSUM52PAM250 ~ BLOSUM45 Highly Divergent

Query length Matrix Gap costs

<35 PAM30 9,1

35-50 PAM70 10,1

50-85 BLOSUM80 10,1

>85 BLOSUM62 11,1

Use Recommendations

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ExampleQuery: >ADRM1_HUMAN

(Proteasomal ubiquitin receptor)Data Base: nr on Human genome.Blast Program: BLASTPMatrices: PAM30,BLOSUM45

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PAM 30 BLOSUM45

•With BLOSUM45 we found related and divergent sequences.

•With PAM30 we found only related sequences.

What difference do we observe?

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PAM 30

BLOSUM45

With BLOSUM45 we can discover interesting relations between proteins

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Mucin-13:a glycosylated membrane protein that protects the cell by binding to pathogens

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With PAM 30

With BLOSUM45

Using different scoring matrices can produce slightlyDifferent alignments:

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A same alignment can be solved in many ways, specially when using a matrix for highly divergent sequences (BLOSUM45):

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PSI-BLAST

Position Specific Iterative BLAST

We will analyze the following Archeal uncharacterized protein: >gi|2501594|sp|Q57997|Y577_METJA PROTEIN MJ0577MSVMYKKILYPTDFSETAEIALKHVKAFKTLKAEEVILLHVIDEREIKKRDIFSLLLGVAGLNKSVEEFENELKNKLTEEAKNKMENIKKELEDVGFKVKDIIVVGIPHEEIVKIAEDEGVDIIIMGSHGKTNLKEILLGSVTENVIKKSNKPVLVVKRKNS

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Threshold for initial BLAST

Search (default:10)

Threshold for inclusion in PSI-BLAST iterations

(default:0.005)

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The query itself

Orthologous sequences in two other archaeal species

Other homologous sequences

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...

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Is MJ0577 a filament protein?

Is MJ0577 a cationic amino

transporter?

Is MJ0577 a universal stress

protein?22