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. Automated map interpretation Fit amino acids into density in the right orientation Largely manual process –Molecular graphics programs –Bottleneck step: time consuming & error-prone Diffraction data is typically of poor quality –Focus of TEXTAL ™ : medium-poor resolution Modeling requires a lot of experience –Automation is very challenging; often considered an art! Other automated model building programs: ARP/wARP, RESOLVE, MAIN –Other AI approaches: expert system, molecular scene analysis

5. 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) Overview of TEXTAL Electron density map (or structure factors) TEXTAL Protein model (may need refinement)

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

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

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

9. 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 C  ’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

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

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

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

13. 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.

14. F=

15. 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,{R i },matches,mismatches) choose feature f  F at random for each, R j  matches(R i ),R k  mismatches(R i ) compute cross-over point i where: dist’(R i,R j )=dist’(R i,R k ) dist’(X,Y)= (X f -Y f ) 2 +(1- )dist \f (X,Y) pick that is best compromise among i ranks most matches above mismatches update weight vector: w’  update(w,f, ), w f ’= repeat until convergence

16. SLIDER Results

17. Stage 3: Post-Processing

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

19. 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,  fold Example of density quality (~1  contour with C  trace)

20. Electron density map (2.8A)

21. Results of tracing

23. Strip off branches of trace (linearize)

24. Linearized trace shows backbone connectivity

25. Pick C  ’s using neural net; link together

26. Results of CAPRA

27. 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

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

29. Results of LOOKUP (modeling side-chains)

30. 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

31. Closeup of  -strand (TEXTAL model in green)

32. Closeup of another  -strand and turn

33. 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

34. Deployment September 2004: Linux and OSX distributions –Can be downloaded from http://textal.tamu.edu:12321http://textal.tamu.edu:12321 –40 trial licenses granted so far June 2002: WebTex (http://textal.tamu.edu:12321)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

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

36. 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

37. 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)