1. NIGMS Protein Structure Initiative: Target Selection Workshop ADDA and remote homologue detection Liisa Holm Institute of Biotechnology University of Helsinki

2. Definitions Nrdbxx = nrdb where no two sequences are more than xx % identical; redundant sequences are mapped to representative –Uniprot + Genpept + PIR + PDB + … –Nrdb100 – Nrdb90 – … – Nrdb40 – Nrdb30 = “modeling family” PairsDB = database of all-against-all comparisons –Blast in nrdb90, PSI-Blast in nrdb40 BIG = family detected by profile comparison –Profile needs seed set (alignment); automatic iterative profile construction has poor convergence –Profiles  Partially overlapping neighbour sets  Need to cluster sequences  Clustering artefacts when true cluster shape is non-spherical

3. (graph) covering ≠ clustering ≠ classification Incomplete detection of homologous set by profile models Example: Urease et al. superfamily IDEAL REAL

4. ADDA: clustering of domains into families ADDA = Automatic Domain Definition Algorithm –Heger & Holm (2003) J Mol Biol 328, 749-767. –Heger & al (2005) Nucl. Acids Res. 33 Database Issue, D188-D191. Principles of ADDA –Blast all-against-all comparison in nrdb90 –Domains are optimally covered by alignments Complete domain coverage; every residue belongs to a domain –Minimum/maximum spanning tree of domains –Remove links where profile-profile score is below threshold –Connected components are domain families Quality assessment –Most ADDA families are pure, containing one PFAM family or SCOP superfamily (plus previously unclassified members) –Occasionally members from different PFAM family are merged in one ADDA family (contamination or PFAM misclassification) –Domain size distribution is reasonable For example, much less over-fragmentation than by Prodom algorithm

5. ADDA purity and domain size PFAM SCOP Accuracy of domain boundaries -Red: best possible in domain tree -Black: actually selected

6. 3D coverage of model proteomes PDB entries from May 2006 –Required greater than 80 % overlap between PDB sequence and ADDA domain to call family structurally covered ADDA domain families –BIG families 28429 families have more than ten members in nrdb100 –2383 structurally covered BIG families 8820 families have more than ten members in nrdb40 –1869 structurally covered BIG families NCBI genome sets –H sapiens, C elegans, D melanogaster, A thaliana, E coli, B anthracis, T maritima –Mapped to ADDA families 6770 BIG(nrdb40) families occur in model genome set –1705 structurally covered

7. Model genome coverage – 28429 BIG families in nrdb100 T. maritima would be covered by 1000 BIG families and is two thirds done

8. 6770 BIG families in nrdb40 Multigene families in eukaryotes 2.0-2.4 domains per euk. gene; 1.3 domains per prok. gene

9. Seven model genomes HumanWorm, fly, plant Prokaryotes (E coli, B anthracis, T maritima) 6382412 29 187 802 161 836 Human BIG target families are almost exclusively eukaryote- specific 57535 18 588 88 264 155 HumanWorm, fly, plant Prokaryotes Universal BIG families are almost covered 5065 white BIG target families 1705 structurally covered BIG families

10. Covering all modelling families will have astronomical cost Nrdbxx updates; Nrdb30 = “modelling family”

11. Fine-grained coverage MF: Structural core shrinks rapidly below 30 % sequence identity –  Need less naïve modelling software capable of building those parts ab initio which are not covered by template –Misalignment is major source of error  Transitive alignment covers more of the structurally equivalent core Average coverage of structural core (152 pairs in 11 superfamilies): Transitive 51 % Global alignment (HMMer) 43 % Local alignment (PSI-Blast) 34 % ErrorRmsd/A Template32 Misaligned16 Loops8 Backbone4 Rotamers2

12. Coarse-grained coverage BIG/BIGGER: Homology detection –Difficulty of aligning remote homologues  Shared sequence motifs suggest conserved biochemical mechanism  Functional classification –Sequence comparison only detects half of remote homologue pairs  Structure comparison reveals missing links Transitive search for conserved motifs detects more remote homologues than profile-profile comparison

13. Clustering PFAM families Comparison of ADDA to PFAM-A resulted in extension but no discovery of completely new large families PFAM-A v.19: 7340 families, 2451 covered according to PFAM’s assignments, 1396 families in 205 clans Our method achieved 30 % coverage of clan relationships at 5 % error rate compared to 23 % coverage at 5 % error rate by profile-profile comparison –1083 unclassified PFAMs linked to 205 known clans 1219 white PFAMs linked to known structure in 155 clusters –1256 PFAMs clustered in 470 predicted clans 336 white PFAMs linked to known structure in 222 clusters –3610 PFAMS remained singletons 2352 white PFAMs  2451 covered, ~1555 fold assignments, ~3334 targets

14. Conclusions ADDA  ~3000 human target families –~40 % reduction in number of PFAM target families by fold assignment (based on sequence only) Coarse-grained coverage yields information out of reach to sequence comparison –Need to improve measures of sequence similarity to infer homology Sequence motif-based functional classification –Need to increase the radius of convergence in template- based structure prediction Protein complexes  hypothesis-driven research –Large conformational changes –Multigene receptor-ligand pair discrimination involves rotations in docking orientation

15. Acknowledgements Andreas Heger, Oxford University Swapan Mallick, Ashwin Sivakumar, Chris Wilton, Institute of Biotechnology Funding: Academy of Finland, Sigrid Juselius Foundation, EU