Psi-Blast
description
Transcript of Psi-Blast
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Psi-Blast
Morten Nielsen,Department of systems
biology, DTU
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Objectives• Understand why BLAST often fails for
low sequence similarity• See the beauty of sequence profiles
– Position specific scoring matrices (PSSMs)• Use BLAST to generate Sequence
profiles• Use profiles to identify amino acids
essential for protein function and structure
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Sequence ProfilesAlignments based on conventional scoring
matrices (BLOSUM62) scores all positions in a sequence in an equal manner
Some positions are highly conserved, some are highly variable (more than what is described in the BLOSUM matrix)
Sequence profile are ideal suited to describe such position specific variations
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What goes wrong when Blast fails?• Conventional sequence alignment uses a
(Blosum) scoring matrix to identify amino acids matches in the two protein sequences
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Alignment scoring matrices• Blosum62 score matrix. Fg=1. Ng=0?
L A G D S DFIGDSL
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Alignment scoring matrices• Blosum62 score matrix. Fg=1. Ng=0?
• Score =2-1+6+6+4=17
L A G D S DF 0 -2 -3 -3 -2 -3I 2 -1 -4 -3 -2 -3G -4 0 6 -1 0 -1D -4 -2 -1 6 0 6S -2 1 0 0 4 0L 4 -1 -4 -4 -2 -4
LAGDSI-GDS
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What goes wrong when Blast fails?• Conventional sequence alignment uses a
(Blosum) scoring matrix to identify amino acids matches in the two protein sequences• This scoring matrix is identical at all positions
in the protein sequence!EVVFIGDSLVQLMHQC
X X X
X X X
AGDS.GGGDS
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When Blast works!
1PLC
._
1PLB._
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When Blast fails!
1PLC
._
1PMY._
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Sequence profiles
• In reality not all positions in a protein are equally likely to mutate• Some amino acids (active cites) are highly
conserved, and the score for mismatch must be very high
• Other amino acids can mutate almost for free, and the score for mismatch should be lower than the BLOSUM score
• Sequence profiles can capture these differences
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Sequence profiles
TKAVVLTFNTSVEICLVMQGTSIV----AAESHPLHLHGFNFPSNFNLVDGMERNTAGVP
TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADPPAYVTQCPI
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ADDGSLAFVPSEF--SISPGEKIVFKNNAGFPHNIVFDEDSIPSGVDASKISMSEEDLLN TVNGAI--PGPLIAERLKEGQNVRVTNTLDEDTSIHWHGLLVPFGMDGVPGVSFPG---I-TSMAPAFGVQEFYRTVKQGDEVTVTIT-----NIDQIED-VSHGFVVVNHGVSME---IIE--KMKYLTPEVFYTIKAGETVYWVNGEVMPHNVAFKKGIV--GEDAFRGEMMTKD----TSVAPSFSQPSF-LTVKEGDEVTVIVTNLDE------IDDLTHGFTMGNHGVAME---VASAETMVFEPDFLVLEIGPGDRVRFVPTHK-SHNAATIDGMVPEGVEGFKSRINDE----TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADPPAYVTQCPI
Sequence profiles
Conserved
Non-conserved
Matching any thing but G => large negative score
Any thing can match
TKAVVLTFNTSVEICLVMQGTSIV----AAESHPLHLHGFNFPSNFNLVDGMERNTAGVP
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Visualization of Sequence logos
ALAKAAAAMALAKAAAANALAKAAAARALAKAAAATALAKAAAAVGMNERPILTGILGFVFTMTLNAWVKVVKLNEPVLLLAVVPFIVSV
PA = 6/10 = 0.6PG = 2/10 = 0.2PT = PK = 1/10 = 0.1PC = PD = …PV = 0.0
qA = 0.07qG = 0.07qT = 0.05qK = 0.06
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Sequence logos (Kullback-Leibler)
• Height of a column equal to I• Relative height of a letter is p• (Letters upside-down if pa < qa)
High information positions
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ADDGSLAFVPSEF--SISPGEKIVFKNNAGFPHNIVFDEDSIPSGVDASKISMSEEDLLN TVNGAI--PGPLIAERLKEGQNVRVTNTLDEDTSIHWHGLLVPFGMDGVPGVSFPG---I-TSMAPAFGVQEFYRTVKQGDEVTVTIT-----NIDQIED-VSHGFVVVNHGVSME---IIE--KMKYLTPEVFYTIKAGETVYWVNGEVMPHNVAFKKGIV--GEDAFRGEMMTKD----TSVAPSFSQPSF-LTVKEGDEVTVIVTNLDE------IDDLTHGFTMGNHGVAME---VASAETMVFEPDFLVLEIGPGDRVRFVPTHK-SHNAATIDGMVPEGVEGFKSRINDE----TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADGPAYVTQCPI
Sequence profiles
Conserved
Non-conserved
Matching any thing but G => large negative score
Any thing can match
TKAVVLTFNTSVEICLVMQGTSIV----AAESHPLHLHGFNFPSNFNLVDPMERNTAGVP
TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADGPAYVTQCPI
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How to make sequence profiles 1. Align (BLAST) sequence against large
sequence database (Swiss-Prot)2. Select significant alignments and make
sequence profile3. Use profile to align against sequence
database to find new significant hits4. Repeat 2 and 3 (normally 3 times!)
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Protein world
Blast iterations
Protein
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How to make sequence profiles The blast command
blastpgp -d db -e 0.00001 -j 4 -Q blastprofile -i fastafile -o outLast position-specific scoring matrix computed A R N D C Q E G H I L K M F P S T W Y V 1 T -1 -3 -2 -3 -3 -2 -2 -3 -3 -3 -4 -2 -3 -4 -3 5 6 -5 -4 -3 2 T -3 1 -1 -4 -4 -2 -3 -4 -4 -4 -4 5 -4 -5 -4 -2 6 -5 -4 -3 3 V -3 -4 -6 -6 -4 -5 -6 -5 -6 6 2 -5 -2 -2 -6 -5 -3 1 -4 4 4 Y 0 -5 -6 -6 1 -3 -5 -2 -1 0 2 -5 -2 4 -6 -4 -4 0 4 4 5 L -2 -5 -6 -6 3 -5 -6 -6 -5 3 4 -5 1 5 -6 -5 -1 -1 -1 1 6 A 3 -5 -5 -6 0 -2 -5 -3 -5 3 1 -5 1 4 -5 -4 -1 -1 -2 2 7 G -3 -5 -3 -4 -6 -5 -5 7 -5 -7 -7 -5 -6 -6 -5 -2 -4 -6 -6 -6 8 D -3 -4 1 8 -6 -3 -1 -4 -4 -6 -6 -4 -6 -6 -4 -3 -3 -7 -6 -6 9 S -2 -4 -1 -3 -4 -3 -3 -3 -4 -5 -6 -3 -5 -6 -4 7 -2 -6 -5 -5 10 T -2 -3 -4 -5 -4 -3 -5 -5 -5 6 1 -4 -2 -4 -5 -3 5 -5 -4 0 11 M 0 -4 -3 -4 -4 -4 -3 -4 -5 -1 -2 -4 3 -2 -2 1 6 -5 -4 2 12 A 4 -1 0 0 1 2 0 -2 2 -4 -4 -1 -3 -1 -3 0 -1 1 -1 -2
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Sequence profiles for a single sequence
TKAVVLTFNTSVEICLVMQGTSIV----AAESHPLHLHGFNFPSNFNLVDPMERNTAGVP
A R N D C Q E G H I L K M F P S T W Y VA 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
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Sequence profiles (1K7C.A)
0 iterations (Blosum62) A R N D C Q E G H I L K M F P S T W Y VA 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
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Sequence profiles (1K7C.A)
0 iterations (Blosum62) A R N D C Q E G H I L K M F P S T W Y VA 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
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Sequence profiles (1K7C.A)
3 iterations
A R N D C Q E G H I L K M F P S T W Y VT -1 -2 -1 -2 -2 -2 -2 -3 -3 -2 -3 -2 -2 -4 -2 2 6 -4 -3 -1 T -2 3 -2 -3 -3 -2 -3 -4 -3 -3 -4 2 -3 -4 -3 -1 6 -5 -4 -3 V -3 -5 -6 -6 -3 -5 -5 -6 -5 6 2 -5 -1 0 -5 -4 -3 -5 -3 4 Y -3 -5 -5 -6 -4 -4 -5 -3 1 -1 0 -5 -2 6 -5 -4 -4 2 6 2 L -4 -5 -6 -6 1 -5 -5 -6 -5 4 5 -5 1 1 -5 -5 -2 0 -3 1 A 4 -4 -4 -5 2 -4 -4 -2 -5 3 -1 -4 -2 1 -4 -2 -1 -5 -3 2 G -2 -5 -3 -4 -5 -4 -4 7 -4 -6 -6 -4 -5 -5 -4 -1 -4 -5 -5 -5 D -4 -4 0 8 -6 -3 -1 -4 -3 -6 -6 -3 -5 -6 -4 -3 -3 -7 -5 -6 S -1 -3 -2 -3 -3 -2 -2 -3 -3 -5 -5 -3 -4 -5 -3 7 -1 -5 -4 -4 T -3 -4 -3 -4 -3 -3 -3 -4 -4 2 -1 -3 -3 -4 -4 -1 7 -5 -4 -2 M 2 -4 -4 -4 -3 -3 -4 -4 -4 -1 -2 -4 5 -3 -4 -1 3 -5 -4 4 A 5 -2 -1 -1 3 0 -2 -1 -3 -4 -4 -1 -3 -4 -3 1 -2 -4 0 -3 K -1 2 1 3 -4 0 0 0 -3 -2 -4 2 -3 -5 -2 -1 2 -5 -4 1 N -2 -3 3 2 2 1 0 -1 -2 -4 -3 -1 -3 -1 -4 -1 -1 -2 6 -4 G -2 -3 -1 -1 -4 1 0 4 -3 -4 -3 -1 -4 -4 3 1 -3 -4 1 -3 G 0 -2 2 3 -3 0 0 3 1 -3 -2 0 -3 -2 -1 0 0 -3 0 -3 G 1 1 3 -1 -3 -1 0 1 2 -3 -2 0 -3 -3 -1 1 0 0 1 -2 S 2 2 3 1 -3 -2 0 1 0 -3 -3 -1 -3 -3 -3 0 -1 -3 2 -3 G 0 -3 -1 -3 -3 -3 -3 2 -3 -3 -1 -1 -3 -4 6 -1 -2 -4 -3 -1 T -1 1 -2 -2 -3 3 2 -2 -2 -1 -1 1 2 -1 0 -1 1 2 2 -2
Sequence profile
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Example.
>1K7C.A TTVYLAGDSTMAKNGGGSGTNGWGEYLASYLSATVVNDAVAGRSARSYTREGRFENIADVVTAGDYVIVEFGHNDGGSLSTDNGRTDCSGTGAEVCYSVYDGVNETILTFPAYLENAAKLFTAKGAKVILSSQTPNNPWETGTFVNSPTRFVEYAELAAEVAGVEYVDHWSYVDSIYETLGNATVNSYFPIDHTHTSPAGAEVVAEAFLKAVVCTGTSLKSVLTTTSFEGTCL
• What is the function• Where is the active site?
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What would you do?
• Function• Run Blast against PDB• No significant hits
• Run Blast against NR (Sequence database)• Function is Acetylesterase?
• Where is the active site?
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Example. Where is the active site?
1WAB Acetylhydrolase
1G66 Acetylxylan esterase
1USW Hydrolase
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When Blast fails!
1K7A
.A
1WAB._
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Example. (SGNH active site)
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Example. Where is the active site?• Sequence profiles might show you where to
look!• The active site could be around• S9, G42, N74, and H195
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Profile-profile scoring matrix
1K7C
.A
1WAB._
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SummarySequence logo is a power tool to visualize
(binding) motifs– Information content identifies essential residues
for function and/or structural stabilityWeight matrices and sequence profiles can be
derived from very limited number of data using the techniques of– Sequence weighting– Pseudo counts
Weight matrices and sequences profiles can accurately describe binding motifs, sequence conservation, active sites...