Pfam a resource for remote homology domain identification

1. Pfam a resource for remote homology domain identification http://pfam.xfam.org Finn et al NAR 2014

2. Building families Identify target Abandon Build SEED MSA of representative members Build Profile-HMM Search UniProtKB Abandon QCs and fix Significance thresholds Annotate EMBO Workshop, Cape Town, 2014

3. QC: family overlaps Old Family New Family EMBO Workshop, Cape Town, 2014

4. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN EMBO Workshop, Cape Town, 2014

5. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are evolutionary related nature overlaps, profile-profile, functional residues, functional annotation, structure EMBO Workshop, Cape Town, 2014

6. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are evolutionary related • Solution 1: Merge EMBO Workshop, Cape Town, 2014

7. QC: family overlaps Clan Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are evolutionary related • Solution 2: Create/Add to clan EMBO Workshop, Cape Town, 2014

8. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are NOT evolutionary related -> then overlaps might be false positives EMBO Workshop, Cape Town, 2014

9. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are NOT evolutionary related • Solution 1: Separate • (expunge seqs from SEED, trim ends, raise threshold)

10. QC: family overlaps Old Family New Family SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN A – Old and New family are NOT evolutionary related • Solution 2: Manually Edit • (no change to family but sequence removed)

11. False positive detection • Overlaps • Hits Score vs Taxonomic distribution • Known annotation (e.g. functional/structural residues) • Known structures • … EMBO Workshop, Cape Town, 2014

12. Building families Identify target Abandon Build SEED MSA of representative members Build Profile-HMM Search UniProtKB Abandon QCs and fix Significance thresholds Annotate EMBO Workshop, Cape Town, 2014

13. Are all Pfamfamilies structural domains? EMBO Workshop, Cape Town, 2014

14. Pfam families with/without PDB structure PDB (43%) No PDB (57%) EMBO Workshop, Cape Town, 2014

15. Pfamtypes Family Domain Repeat Motif EMBO Workshop, Cape Town, 2014

16. Domain and repeats A B • A - Domain • B - Metal stabilised domain • C - 7 repeats form domain • D - 9 repeats form domain could be unlimited number C D EMBO Workshop, Cape Town, 2014

17. Motifs Example: Lipoprotein attachment site, LPAM_1 Alignment coloured by Residue-type EMBO Workshop, Cape Town, 2014

18. Pfamtypes Family Domain Repeat Disordered Family? EMBO Workshop, Cape Town, 2014

22. PDBid: 2JGC

23. The Pfam website EMBO Workshop, Cape Town, 2014

24. The Pfam website EMBO Workshop, Cape Town, 2014

25. The Pfam website EMBO Workshop, Cape Town, 2014

26. The Pfam website

27. The Pfam website EMBO Workshop, Cape Town, 2014

28. The Pfam website

29. Pfam families’ interactions: iPfam Finn et al. NAR 2013 http://www.ipfam.org

30. Some caveats • Identifying repeats is challenging, especially with HMMER3 ->local • Functional diversity within families and clans • Domains of Unknown Function • Family boundaries if no structure available TUM, January 2013 EMBO Workshop, Cape Town, 2014

31. Comparison of Enolase clan/superfamily in Pfam and SFLD Picture courtesy of Patsy Babbit (UCSF) TUM, January 2013 SFLD: Akiva et al. NAR 2013

32. How far from covering the sequence space: H. sapiens from the Pfam blog: at http://xfam.wordpress.com/tag/pfam/ EMBO Workshop, Cape Town, 2014

33. Building a Pfam family EMBO Workshop, Cape Town, 2014

34. Pick a target region 1. OPEN Chimera 2. File -> Open “2KX7.pdb” 2KX7 TUM, January 2013 EMBO Workshop, Cape Town, 2014

35. Pick a target region 1. Actions-> Ribbon-> hide 2. SELECT “2KX7.pdb (#0.1) chain A” 3. Actions -> Ribbon -> show 2KX7 model 1 TUM, January 2013 EMBO Workshop, Cape Town, 2014

36. Rcs-signaling system bacterial two component system (sensor kinase +response regulator) 2KX7 TUM, January 2013 Schmöe et al. Structure 2011 EMBO Workshop, Cape Town, 2014

37. Pick a target region Look-up UniprotKB ID: P39838 on the Pfam website (http://pfam.xfam.org) TUM, January 2013 EMBO Workshop, Cape Town, 2014

38. Pick a target region Look-up UniprotKB ID: P39838 on the Pfam website (http://pfam.xfam.org) TUM, January 2013 EMBO Workshop, Cape Town, 2014

39. Pick a target region S HK ABL 2KX7 HPt TUM, January 2013 Schmöe et al. Structure 2011 EMBO Workshop, Cape Town, 2014

40. Pick a target region S HK ABL 2KX7 HPt TUM, January 2013 Schmöe et al. Structure 2011 EMBO Workshop, Cape Town, 2014

41. Pick a target region EMBO Workshop, Cape Town, 2014

42. Pick a target region EMBO Workshop, Cape Town, 2014

43. Look for homologs Click Start http://hmmer.janelia.org HMMER website: Finn et al. NAR 2011 EMBO Workshop, Cape Town, 2014

44. Look for homologs http://hmmer.janelia.org Choose “Marco-Data/Other/2KX7.fasta” EMBO Workshop, Cape Town, 2014

45. Select your dataset Select rp75 in Sequence Database EMBO Workshop, Cape Town, 2014

46. Parse hits EMBO Workshop, Cape Town, 2014

47. Parse hits Click EMBO Workshop, Cape Town, 2014

48. Check conservation and coverage EMBO Workshop, Cape Town, 2014

49. Check low scores Scroll down EMBO Workshop, Cape Town, 2014

50. Check taxonomic distribution Click Taxonomy EMBO Workshop, Cape Town, 2014