TEXTAL - Automated Crystallographic Protein Structure Determination Using Pattern Recognition

1. TEXTAL - Automated Crystallographic Protein Structure Determination Using Pattern Recognition Principal Investigators: Thomas Ioerger (Dept. Computer Science) James Sacchettini (Dept. Biochem/Biophys) Other contributors: Tod D. Romo, Kreshna Gopal, Erik McKee, Lalji Kanbi, Reetal Pai & Jacob Smith Funding: National Institutes of Health Texas A&M University

2. X-ray crystallography • Most widely used method for protein modeling • Steps: • Grow crystal • Collect diffraction data • Generate electron density map (Fourier transform) • Interpret map i.e. infer atomic coordinates • Refine structure • Model-building • Currently: crystallographers • Challenges: noise, resolution • Goal: automation

3. X-ray crystallography • Most widely used method for protein modeling • Steps: • Grow crystal • Collect diffraction data • Generate electron density map (Fourier transform) • Interpret map i.e. infer atomic coordinates • Refine structure • Model-building • Currently: crystallographers • Challenges: noise, resolution • Goal: automation

4. Overview of TEXTAL • Automated model-building program • Can we automate the kind of visual processing of patterns that crystallographers use? • Intelligent methods to interpret density, despite noise • Exploit knowledge about typical protein structure • Focus on medium-resolution maps • optimized for 2.8A (actually, 2.6-3.2A is fine) • typical for MAD data (useful for high-throughput) • other programs exist for higher-res data (ARP/wARP) Electron density map (or structure factors) Protein model (may need refinement) TEXTAL

5. Crystal Collect data Electron density map Diffraction data LOOKUP: model side chains CAPRA: models backbone SCALE MAP TRACE MAP CALCULATE FEATURES PREDICT Cα’s BUILD CHAINS PATCH & STITCH CHAINS REFINE CHAINS Model of backbone Model of backbone & side chains POST-PROCESSING SEQUENCE ALIGNMENT REAL SPACE REFINEMENT Corrected & refined model

6. CAPRA: C-Alpha Pattern-Recognition Algorithm tracing • Best-first search with heuristic • scoring function based on: • neural net scores • density • connectivity • secondary structure Neural network: estimates which pseudo-atoms are closest to true Ca’s linking

7. Example of Ca-chains fit by CAPRA Rat a2 urinary protein (P. Adams) data: 2.5A MR map generated at 2.8A % built: 84% # chains: 2 lengths: 47, 88 RMSD: 0.82A

8. Stage 2: LOOKUP • LOOKUP is based on Pattern Recognition • Given a local (5A-spherical) region of density, have we seen a pattern like this before (in another map)? • If so, use similar atomic coordinates. • Use a database of maps with known structures • 200 proteins from PDB-Select (non-redundant) • back-transformed (calculated) maps at 2.8A (no noise) • regions centered on 50,000 Ca’s • Use feature extraction to match regions efficiently • feature (e.g. moments) represent local density patterns • features must be rotation-invariant (independent of 3D orientation) • use density correlation for more precise evaluation

9. BUILD CHAINS: Examines network of Cα’s and use heuristic search to connect them to form backbone chains CAPRA

10. LOOKUP: Uses case-based reasoning to find, for each Cα, the best matching local region in a database

11. The LOOKUP Process Find optimal rotation Database of known maps Two-step filter: 1) by features 2) by density correlation “2-norm”: weighted Euclidean distance metric for retrieving matches: Region in map to be interpreted

12. Examples of Numeric Density Features • Distance from center-of-sphere to center-of-mass • Moments of inertia - relative dispersion along orthogonal axes • Geometric features like “Spoke angles” • Local variance and other statistics Features are designed to be rotation-invariant, i.e. same values for region in any orientation/frame-of-reference. TEXTAL uses 19 distinct numeric features to represent the pattern of density in a region, each calculated over 4 different radii, for a total of 76 features.

13. F=<1.72,-0.39,1.04,1.55...> F=<1.58,0.18,1.09,-0.25...> F=<0.90,0.65,-1.40,0.87...> F=<1.79,-0.43,0.88,1.52...>

14. SLIDER: Feature-weighting algorithm • Euclidean distance metric used for retrieval: • importance of relevant features, avoid noisy features • Goal: find optimal weight vector w the generates highest probability of hits (matches) in top K candidates from database • Concept of Slider: • analyze distances between representative matches and mismatches • adjust features so the most matches are ranked higher than mismatches Slider Algorithm(w,F,{Ri},matches,mismatches) choose feature fF at random for each <Ri,Rj,Rk>, Rjmatches(Ri),Rkmismatches(Ri) compute cross-over point li where: dist’(Ri,Rj)=dist’(Ri,Rk) dist’(X,Y)= l(Xf-Yf)2+(1-l)dist\f(X,Y) pick l that is best compromise among li ranks most matches above mismatches update weight vector: w’update(w,f,l), wf’=l repeat until convergence

15. SLIDER Results

16. Stage 3: Post-Processing

17. Quality of TEXTAL models • Typically builds >80% of the protein atoms • Accuracy of coordinates: ~1Å error (RMSD) • Depends on resolution and quality of map

18. PcaA • Mycolic acid cyclopropyl synthase (Smith&Sacchettini) • original structure solved at 2.0A via MAD R-value = 0.22, R-free = 0.27 • 287 residues, a/b fold Example of density quality (~1s contour with Ca trace)

19. Electron density map (2.8A)

20. Results of tracing

22. Strip off branches of trace (linearize)

23. Linearized trace shows backbone connectivity

24. Pick Ca’s using neural net; link together

25. Results of CAPRA

26. Comparison to backbone of true structure (white) Percent built = 89% (missing: 15-residue N-terminus, 17-residue disordered loop) 4 single-atom insertions; 5 single-atom deletions RMSD = 0.81A

27. CAPRA model consists of 3 chains Chain lengths: 14, 96, 145 residues

28. Results of LOOKUP (modeling side-chains)

29. Comparison of TEXTAL model to true structure Percent amino acid identity = 87.5% (mistakes: small frame-shifts around gaps in alignment) all-atom RMSD = 0.92A

30. Closeup of b-strand (TEXTAL model in green)

31. Closeup of another b-strand and turn

32. Implementation • Project started in 1998 • Collaboration between TAMU Computer Science & Biochemistry departments • 100,000 lines of C/C++, Perl, Python code • ~8 developers • CVS for version management • Platforms: Irix, Linux, OSX, Win32 • Speed: 1-3 hours for medium-sized proteins

33. Deployment • September 2004: Linux and OSX distributions • Can be downloaded from http://textal.tamu.edu:12321 • 40 trial licenses granted so far • June 2002: WebTex (http://textal.tamu.edu:12321) • Till May 2005: TB Structural Genomics Consortium members only • Recently open to the public • ~500 jobs successfully processed • 120 users from 70 institutions in 20 countries • July 2003: Model building component of PHENIX • Python-based Hierarchical ENvironment for Integrated Xtallography • Consortium members: • Lawrence Berkeley National Lab • University of Cambridge • Los Alamos National Lab • Texas A&M University • April 2005: Alpha release - over 300 downloads so far

34. Python-based Hierarchical ENvironment for Integrated Xtallography HYSS, CCTBX (Lawrence Berkeley Lab) Crystallography toolbox, heavy atom search, refinement PHENIX diffraction data PHASER (University of Cambridge) Maximum likelihood phasing SOLVE/RESOLVE (Los Alamos National Lab) Statistical density modification, minimum bias phasing refined molecular model TEXTAL™ (Texas A&M University) Model building

35. Conclusions • Pattern recognition is a successful technique for macromolecular model-building • Future directions: • recognizing disulfide bridges, metal ions, detergents... • building ligands, co-factors, etc. • using models built to iteratively improve phases • building at higher or lower resolutions • intelligent agent for guiding model-completion • detecting and exploiting non-crystallographic symmetry • building nucleic acids (RNA and DNA) • Importance and challenges of interdisciplinary research

36. Acknowledgements • Funding: • National Institutes of Health • Our group: • Jacob Smith, Kreshna Gopal, Lalji Kanbi, Erik McKee, Reetal Pai, Tod Romo • Our association with the PHENIX group: • Paul Adams (Lawrence Berkeley National Lab) • Randy Read (Cambridge University) • Tom Terwilliger (Los Alamos National Lab)