1. Morten Nielsen, CBS, BioCentrum, DTU
Psi-Blast
Morten Nielsen,
CBS, BioCentrum,
DTU

2. Understand why BLAST often fails for low sequence similarity
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

3. What goes wrong when Blast fails?
Conventional sequence alignment uses a (Blosum) scoring matrix to identify amino acids matches in the two protein sequences

4. Blosum scoring matrix A R N D C Q E G H I L K M F P S T W Y V

5. Alignment scoring matrices
Blosum62 score matrix. Fg=1. Ng=0? L A G D S F I

6. Alignment scoring matrices
Blosum62 score matrix. Fg=1. Ng=0?
Score = =17 L A G D S F -2 -3 I 2 -1 -4 6 1 4
LAGDS
I-GDS

7. 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
AGDS.GGGDS

8. When Blast works!
1PLC._
1PLB._

9. When Blast fails!
1PLC._
1PMY._

10. When Blast fails

11. 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

12. What are sequence profiles?

13. Binding Motif. MHC class I with peptide
Anchor positions

14. Sequence information
SLLPAIVEL YLLPAIVHI TLWVDPYEV GLVPFLVSV KLLEPVLLL LLDVPTAAV LLDVPTAAV LLDVPTAAV
LLDVPTAAV VLFRGGPRG MVDGTLLLL YMNGTMSQV MLLSVPLLL SLLGLLVEV ALLPPINIL TLIKIQHTL
HLIDYLVTS ILAPPVVKL ALFPQLVIL GILGFVFTL STNRQSGRQ GLDVLTAKV RILGAVAKV QVCERIPTI
ILFGHENRV ILMEHIHKL ILDQKINEV SLAGGIIGV LLIENVASL FLLWATAEA SLPDFGISY KKREEAPSL
LERPGGNEI ALSNLEVKL ALNELLQHV DLERKVESL FLGENISNF ALSDHHIYL GLSEFTEYL STAPPAHGV
PLDGEYFTL GVLVGVALI RTLDKVLEV HLSTAFARV RLDSYVRSL YMNGTMSQV GILGFVFTL ILKEPVHGV
ILGFVFTLT LLFGYPVYV GLSPTVWLS WLSLLVPFV FLPSDFFPS CLGGLLTMV FIAGNSAYE KLGEFYNQM
KLVALGINA DLMGYIPLV RLVTLKDIV MLLAVLYCL AAGIGILTV YLEPGPVTA LLDGTATLR ITDQVPFSV
KTWGQYWQV TITDQVPFS AFHHVAREL YLNKIQNSL MMRKLAILS AIMDKNIIL IMDKNIILK SMVGNWAKV
SLLAPGAKQ KIFGSLAFL ELVSEFSRM KLTPLCVTL VLYRYGSFS YIGEVLVSV CINGVCWTV VMNILLQYV
ILTVILGVL KVLEYVIKV FLWGPRALV GLSRYVARL FLLTRILTI HLGNVKYLV GIAGGLALL GLQDCTMLV
TGAPVTYST VIYQYMDDL VLPDVFIRC VLPDVFIRC AVGIGIAVV LVVLGLLAV ALGLGLLPV GIGIGVLAA
GAGIGVAVL IAGIGILAI LIVIGILIL LAGIGLIAA VDGIGILTI GAGIGVLTA AAGIGIIQI QAGIGILLA
KARDPHSGH KACDPHSGH ACDPHSGHF SLYNTVATL RGPGRAFVT NLVPMVATV GLHCYEQLV PLKQHFQIV
AVFDRKSDA LLDFVRFMG VLVKSPNHV GLAPPQHLI LLGRNSFEV PLTFGWCYK VLEWRFDSR TLNAWVKVV
GLCTLVAML FIDSYICQV IISAVVGIL VMAGVGSPY LLWTLVVLL SVRDRLARL LLMDCSGSI CLTSTVQLV
VLHDDLLEA LMWITQCFL SLLMWITQC QLSLLMWIT LLGATCMFV RLTRFLSRV YMDGTMSQV FLTPKKLQC
ISNDVCAQV VKTDGNPPE SVYDFFVWL FLYGALLLA VLFSSDFRI LMWAKIGPV SLLLELEEV SLSRFSWGA
YTAFTIPSI RLMKQDFSV RLPRIFCSC FLWGPRAYA RLLQETELV SLFEGIDFY SLDQSVVEL RLNMFTPYI
NMFTPYIGV LMIIPLINV TLFIGSHVV SLVIVTTFV VLQWASLAV ILAKFLHWL STAPPHVNV LLLLTVLTV
VVLGVVFGI ILHNGAYSL MIMVKCWMI MLGTHTMEV MLGTHTMEV SLADTNSLA LLWAARPRL GVALQTMKQ
GLYDGMEHL KMVELVHFL YLQLVFGIE MLMAQEALA LMAQEALAF VYDGREHTV YLSGANLNL RMFPNAPYL
EAAGIGILT TLDSQVMSL STPPPGTRV KVAELVHFL IMIGVLVGV ALCRWGLLL LLFAGVQCQ VLLCESTAV
YLSTAFARV YLLEMLWRL SLDDYNHLV RTLDKVLEV GLPVEYLQV KLIANNTRV FIYAGSLSA KLVANNTRL
FLDEFMEGV ALQPGTALL VLDGLDVLL SLYSFPEPE ALYVDSLFF SLLQHLIGL ELTLGEFLK MINAYLDKL
AAGIGILTV FLPSDFFPS SVRDRLARL SLREWLLRI LLSAWILTA AAGIGILTV AVPDEIPPL FAYDGKDYI
AAGIGILTV FLPSDFFPS AAGIGILTV FLPSDFFPS AAGIGILTV FLWGPRALV ETVSEQSNV ITLWQRPLV

15. Sequence Information
Say that a peptide must have L at P2 in order to bind, and that A,F,W,and Y are found at P1. Which position has most information?
How many questions do I need to ask to tell if a peptide binds looking at only P1 or P2?

16. Sequence Information
Say that a peptide must have L at P2 in order to bind, and that A,F,W,and Y are found at P1. Which position has most information?
How many questions do I need to ask to tell if a peptide binds looking at only P1 or P2?
P1: 4 questions (at most)
P2: 1 question (L or not)
P2 has the most information

17. Sequence Information
Say that a peptide must have L at P2 in order to bind, and that A,F,W,and Y are found at P1. Which position has most information?
How many questions do I need to ask to tell if a peptide binds looking at only P1 or P2?
P1: 4 questions (at most)
P2: 1 question (L or not)
P2 has the most information
Calculate pa at each position
Entropy
Information content
Conserved positions
PV=1, P!v=0 => S=0, I=log(20)
Mutable positions
Paa=1/20 => S=log(20), I=0

18. Sequence information - I
ALAKAAAAM
ALAKAAAAN
ALAKAAAAR
ALAKAAAAT
ALAKAAAAV
GMNERPILT
GILGFVFTM
TLNAWVKVV
KLNEPVLLL
AVVPFIVSV
PA = 6/10 = 0.6
PG = 2/10 = 0.2
PT = PK = 1/10 = 0.1
PC = PD = …PV = 0.0
Multiple Sequence alignment

19. Information content
A R N D C Q E G H I L K M F P S T W Y V S I

20. Sequence logos Height of a column equal to I
Relative height of a letter is p
Highly useful tool to visualize sequence motifs
HLA-A0201
High information
positions

21. Sequence logos Relative height of a letter is p
High information
positions
Height of a column equal to I
Relative height of a letter is p
Letters upside-down if pa < qa

22. Protein structure classification
Protein world
Protein superfamily
Protein fold
Protein family

23. Sequence profiles Matching any thing but G => large negative score
Conserved
Non-conserved
ADDGSLAFVPSEF--SISPGEKIVFKNNAGFPHNIVFDEDSIPSGVDASKISMSEEDLLN
TVNGAI--PGPLIAERLKEGQNVRVTNTLDEDTSIHWHGLLVPFGMDGVPGVSFPG---I
-TSMAPAFGVQEFYRTVKQGDEVTVTIT-----NIDQIED-VSHGFVVVNHGVSME---I
IE--KMKYLTPEVFYTIKAGETVYWVNGEVMPHNVAFKKGIV--GEDAFRGEMMTKD---
-TSVAPSFSQPSF-LTVKEGDEVTVIVTNLDE------IDDLTHGFTMGNHGVAME---V
ASAETMVFEPDFLVLEIGPGDRVRFVPTHK-SHNAATIDGMVPEGVEGFKSRINDE----
TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADGPAYVTQCPI
TVNGQ--FPGPRLAGVAREGDQVLVKVVNHVAENITIHWHGVQLGTGWADGPAYVTQCPI
TKAVVLTFNTSVEICLVMQGTSIV----AAESHPLHLHGFNFPSNFNLVDPMERNTAGVP
Matching any thing but G => large negative score
Any thing can match

24. How to make sequence profiles
Align (BLAST) sequence against large sequence database (Swiss-Prot)
Select significant alignments and make sequence profile
Use profile to align against sequence database to find new significant hits
Repeat 2 and 3 (normally 3 times!)

25. Sequence profiles (1J2J.B)
>1J2J.B mol:aa PROTEIN TRANSPORT
NVIFEDEEKSKMLARLLKSSHPEDLRAANKLIKEMVQEDQKRMEK

26. Sequence profiles (1J2J.B)
>1J2J.B mol:aa PROTEIN TRANSPORT
NVIFEDEEKSKMLARLLKSSHPEDLRAANKLIKEMVQEDQKRMEK
A R N D C Q E G H I L K M F P S T W Y V
1 N
2 V
3 I
4 F
5 E
6 D
7 E
8 E
9 K
10 S
11 K
12 M

27. Sequence profiles (1J2J.B)
>1J2J.B mol:aa PROTEIN TRANSPORT
NVIFEDEEKSKMLARLLKSSHPEDLRAANKLIKEMVQEDQKRMEK
Sequence Profile
Blosum62

28. Example. What is the function Where is the active site? >1K7C.A
TTVYLAGDSTMAKNGGGSGTNGWGEYLASYLSATVVNDAVAGRSARSYTREGRFENIADV
VTAGDYVIVEFGHNDGGSLSTDNGRTDCSGTGAEVCYSVYDGVNETILTFPAYLENAAKL
FTAKGAKVILSSQTPNNPWETGTFVNSPTRFVEYAELAAEVAGVEYVDHWSYVDSIYETL
GNATVNSYFPIDHTHTSPAGAEVVAEAFLKAVVCTGTSLKSVLTTTSFEGTCL
What is the function
Where is the active site?

29. Where is the active site?
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?

30. Example. Where is the active site?
1G66 Acetylxylan esterase
1USW Hydrolase
1WAB Acetylhydrolase

31. When Blast fails!
1K7A.A
1WAB._

32. Example. (SGNH active site)

33. 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

34. Profile-profile scoring matrix
1K7C.A
1WAB._

35. Example. Where is the active site?
Align using sequence profiles
ALN 1K7C.A 1WAB._ RMSD = % ID
1K7C.A TVYLAGDSTMAKNGGGSGTNGWGEYLASYLSATVVNDAVAGRSARSYTREGRFENIADVVTAGDYVIVEFGHNDGGSLSTDN
S G N
1WAB._ EVVFIGDSLVQLMHQCE---IWRELFS---PLHALNFGIGGDSTQHVLW--RLENGELEHIRPKIVVVWVGTNNHG------
1K7C.A GRTDCSGTGAEVCYSVYDGVNETILTFPAYLENAAKLFTAK--GAKVILSSQTPNNPWETGTFVNSPTRFVEYAEL-AAEVA
1WAB._ HTAEQVTGGIKAIVQLVNERQPQARVVVLGLLPRGQ-HPNPLREKNRRVNELVRAALAGHP
1K7C.A GVEYVDHWSYVDSIYETLGNATVNSYFPIDHTHTSPAGAEVVAEAFLKAVVCTGTSL H
1WAB._ RAHFLDADPG---FVHSDG--TISHHDMYDYLHLSRLGYTPVCRALHSLLLRL---L

36. Where is the active site?
Rhamnogalacturonan
acetylesterase (1k7c)

37. How to do it? Example >QUERY1
MKDTDLSTLLSIIRLTELKESKRNALLSLIFQLSVAYFIALVIVSRFVRYVNYITYNNLV
EFIIVLSLIMLIIVTDIFIKKYISKFSNILLETLNLKINSDNNFRREIINASKNHNDKNK
LYDLINKTFEKDNIEIKQLGLFIISSVINNFAYIILLSIGFILLNEVYSNLFSSRYTTIS
IFTLIVSYMLFIRNKIISSEEEEQIEYEKVATSYISSLINRILNTKFTENTTTIGQDKQL
YDSFKTPKIQYGAKVPVKLEEIKEVAKNIEHIPSKAYFVLLAESGLRPGELLNVSIENID
LKARIIWINKETQTKRAYFSFFSRKTAEFLEKVYLPAREEFIRANEKNIAKLAAANENQE
IDLEKWKAKLFPYKDDVLRRKIYEAMDRALGKRFELYALRRHFATYMQLKKVPPLAINIL
QGRVGPNEFRILKENYTVFTIEDLRKLYDEAGLVVLE

38. Using Iterative Blast

39. Using Iterative Blast

40. Using Iterative Blast

41. Using Iterative Blast

42. Using Iterative Blast (1st iteration)

43. Using Iterative Blast (3rd iteration)

44. HHpred webserver

45. Take home message
Blast will often fail to recognize sequence relationships for low homology sequence pairs
Sequence profiles contain information on conserved/variable residues in a protein sequence
Sequence profiles are calculated from (multiple) sequence alignments
Iterative Blast enables homology recognition also for low sequence similarity
Sequence profiles give information on residues essential for protein function and protein structure