1. Exercises Pairwise alignment Homology search (BLAST) Multiple alignment (CLUSTAL W) Iterative Profile Search: Profile Search –Pfam –Prosite –PSI-BLAST –SAM

2. Exercises Overview Query Sequence Unknown Blast Sequence to search for close homologs Search pFAM, Prosite for conserved motifs You detected homology with an annotated protein family Make a multiple sequence alignment Generate profile or HMM Search database for remote homologs Blast ClustalW PFAM PROSITE HMMer, PSSM Profile Search PSI-blast

3. Exercises OUT IN Cytc Fe Cu B Fe e-e- I e-e- e-e- O2O2 H2OH2O Terminal Oxidases Unknown protein is a heme cupper oxidase Enzyme that reduces O2 to H2O in respiratory chain Subunit contains 2 hemes and a Cu prosthetic group The residues that are ligands of these groups have been conserved in all types of terminal oxidase complexes

4. Exercises H+H+H+H+ e-e- Nadh.dh succ.dh NADH e-e-e-e- e-e-e-e- succinate O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O e-e-e-e- cytochrome c oxidase ? ? quinol oxidase e-e- H+H+H+H+ cytbc 1 quinol e-e-e-e- Cytc Terminal Oxidases

5. Exercises Multiple Alignment

6. Exercises Multiple alignment: standard gap cost Multiple Alignment Ligands Cu center Ligands hemes Prosite pattern

7. Exercises Multiple alignment: large gap cost Multiple Alignment Ligands Cu center Prosite pattern Ligands hemes

8. Exercises Phylogenetic Tree Tree based on subselection

9. Exercises PROSITE

10. Exercises Prosite

11. Exercises Prosite

12. Exercises Prosite

13. Exercises Prosite

14. Exercises Prosite

15. Exercises

17. Prosite

18. Exercises Prosite domain Prosite

19. Exercises

20. Pattern & profile

21. Exercises

24. pFAM

25. Exercises Pfam

26. Exercises Pfam

27. Exercises Pfam

28. Exercises COX family Pfam

29. Exercises Pfam

30. Exercises Pfam

31. Exercises Pfam

32. Exercises Pfam

33. Exercises Pfam

34. Exercises Pfam

35. Exercises BLOCKS

36. Exercises Blocks

37. Exercises

39. Overview Query Sequence Unknown Blast Sequence to search for close homologs Search pFAM, Prosite for conserved motifs You detected homology with an annotated protein family Make a multiple sequence alignment Generate profile or HMM Search database for remote homologs Blast PFAM PROSITE HMMer, PSSM Profile Search PSI-blast

40. Exercises PSI-BLAST

41. Exercises PSI BLAST –Start from a single sequence –Blast it against NCBI –Select high scoring hits –Perform multiple alignment –Construct profile –Iterate and find remote homologs Usually cut the sequence in pieces Avoid to give as input multi domain proteins PSI-BLAST

42. Exercises PSI-BLAST

43. Exercises PSI-BLAST

44. Exercises PSI-BLAST

45. Exercises PSI-BLAST

46. Exercises PSI-BLAST

47. Exercises PSI-BLAST

48. Exercises SAM

49. Exercises SAM http://www.cse.ucsc.edu/research/compbio/HMM-apps/HMM-applications.html

50. Exercises SAM

51. Exercises SAM

52. Exercises

53. SAM Markov model Emission probability per AA Transition probabilities Insertion probability per AA position short.t2k-w0.5.mod

54. Exercises SAM input targets

55. Exercises SAM

56. Exercises SAM

57. Exercises SAM Hit with highest score! Hit with a protein family for which the 3D structure has been determined

58. Exercises Try to view the structure of the family SAM

59. Exercises SAM

60. Exercises SAM

61. Exercises Logos of the secondary structure prediction SAM

62. Exercises SAM

63. Exercises HMMer states Emission probability per AA Null model Transition probabilities Insertion probability per AA

64. Exercises