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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

22. PDBid: 2JGC

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

24. The Pfam website

25. EMBO Workshop, Cape Town, 2014

26. The Pfam website

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

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

30. TUM, January 2013 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 EMBO Workshop, Cape Town, 2014

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

46. Parse hits EMBO Workshop, Cape Town, 2014

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

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

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

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

51. Check taxonomic distribution EMBO Workshop, Cape Town, 2014

52. Check domain architectures/overlaps EMBO Workshop, Cape Town, 2014 Click Domain

53. Download aligned hits EMBO Workshop, Cape Town, 2014 CLICK on Download and then on Aligned FASTA 1. Save as “RcsD-ABL-hmmer-ali.fasta” 2.

54. OPEN Jalview 1. File -> Input Alignment -> From File “RcsD-ABL-hmmer-ali.fasta” 2. Manipulate alignment