RECOORD RE calculated COOR dinates D atabase

1. RECOORDREcalculated COORdinates Database Jurgen Doreleijers Center for Eukaryotic Structural Genomics University of Madison-Wisconsin jurgen@bmrb.wisc.edu Aart Nederveen Bijvoet Center for Biomolecular Research Utrecht University a.j.nederveen@chem.uu.nl Wim Vranken Macromolecular Structure Database European Bioinformatics Institute wim@ebi.ac.uk

2. Aim • Recalculation of protein structures based on deposited NMR restraints using state of the art methods • Goals: • decrease user- and software-dependent biases • allow a bettercomparison between structures • comparison between different structure calculation programs • provide a database for the development and assessments of validation tools and calculation protocols

3. Overview recalculation project EBI/UU: Generation of consistent STAR files PDB: -coordinates -restraints BMRB: STAR files Doreleijers et al. 2003 2 3 1 restraint manipulation CNS -topology -MD SA -refinement CYANA -sequence -MD SA -… 5 4 recalculation design of RECOORD 6 analysis analysis -improvement? -correlations? -…

4. Databases now publicly available • DOCR/FRED (BMRB) databases containing converted and filtered restraints http://www.bmrb.wisc.edu/servlets/MRGridServlet • RECOORD (EBI) database containing recalculated coordinates http://www.ebi.ac.uk/msd/recoord

5. PDB: -coordinates -restraints BMRB: STAR files Doreleijers et al. 2003 2 1 Selection • Formats (if distance restraints available): • CNS/XPLOR • DIANA/DYANA/CYANA • DISCOVER/MSI • PDB entries selected: • only proteins • no HET atoms • multimers allowed (not yet re-calculated) • at least 20 residues • Finally 545 monomers were selected

6. EBI/UU: Generation of consistent STAR files 3 Conversion issues • Data is converted to formats readable by calculation software (e.g. XPLOR/CNS and CYANA) by the FormatConverter available within CCPN software (Wim Vranken, EBI). Problems: • Differences between coordinate and restraint data: • e.g. 1 chain in pdb entry, 2 chains in restraint list • residue numbering can differ in PDB entry and restraint list • restraints for residues not present in PDB entry… • Nomenclature in restraint list

7. CNS -topology -MD SA -refinement CYANA -sequence -MD SA -… 5 4 Building topology • Starting script: generate_easy.inp from CNS • Automated detection in original ensemble of: • Disulfide bridges (<3Å S-S distance in original first models) • CIS peptides (if |w|<25º in original first models) • Protonation state of histidines (use CNS patches HISD, HISE) • CYANA: sequence based on CNS topology • Add CYSS, HIST, HIST+, cPRO in sequence • Automated generation of disulfide restraints

8. CNS -topology -MD SA -refinement CYANA -sequence -MD SA -… 5 4 CONDOR computer cluster CS University Madison • More than 800 processor used • Total CPU time: 31,169 hours (3.5 years on single workstation) • Example 2EZM, calculation of 1 model (101 a.a. & 2.2 GHz P4 computer) CYANA 31 seconds CNS 340 seconds

9. 6 analysis -improvement? -correlations? -… Evaluation of structure quality • Agreement with experimental restraints • Improvement? • Comparison CNS and CYANA • Relation NMR data quality and structural quality

10. 6 analysis -improvement? -correlations? -… Distance restraints violations ORG: 0.08 Å (0.14 Å) original entries CNW: 0.04 Å (0.05 Å) recalculated in CNS and refined in water frequency RMS distance restraints violations (Å)

11. 6 analysis -improvement? -correlations? -… Dihedral restraints violations ORG: 1.6° (4.6°) original entries CNW: 0.5° (0.5°) recalculated in CNS and refined in water frequency RMS dihedral restraints violations (degrees)

12. 6 analysis -improvement? -correlations? -… Results: quality indicatorsperformance CNS vs. CYANA (no water refinement yet)

13. 6 analysis -improvement? -correlations? -… Results: quality indicatorsperformance CNS before and after water refinement

14. 6 analysis -improvement? -correlations? -… Improvement: packing and Ramachandran Z-scores Improvent Z-score: DZ=Zrefined - Zoriginal For ~ 5 % of entries no improvement possible because of missing NMR data compared to authors improvement Ramachandran missing data improvement packing

15. 6 analysis -improvement? -correlations? -… In search of correlations (Pearson coefficient) refined (correlations higher) original (correlations lower)

16. 6 analysis -improvement? -correlations? -… In search of correlations (Bumps) refined original

17. 6 analysis -improvement? -correlations? -… In search of correlations (NMR data density) refined original

18. 6 analysis -improvement? -correlations? -… Correlation NMR data density Ramachandran Z-score r=0.31 Ramachandran Z-score NMR data density

19. 6 analysis -improvement? -correlations? -… Correlation NOE completeness and packing Z-score r=0.20 NMR data-based indicators cannot yield any indication of the normality of the structures packing Z-score NOE completeness

20. 6 analysis -improvement? -correlations? -… In search of correlations (Precision) refined original

21. 6 analysis -improvement? -correlations? -… Correlation between precision and data density r=-0.46 circular variance NMR data density

22. 6 analysis -improvement? -correlations? -… Correlation between precision and Ramachandran r=-0.67 Protein with high Ramachandran normality will have small circular variance circular variance 1SUT Ramachandran plot appearance (Z-score)

23. 6 analysis -improvement? -correlations? -… Correlation between RMSD and structural uncertainty (QUEEN) r=-0.69 Structural uncertainty imposes lower limit to the RMSD backbone RMSD (Å) structural uncertainty

24. Conclusions I • NMR-STAR files made consistent for 545 out of ±1700 entries • Protocols and scripts available for recalculation in CYANA and CNS • Validation database available for testing of new protocols • Improvement compared to original data: 1 standard deviation closer to X-ray db • violations in original data do no limit recalculation effort • refinement in water required • 5 % no improvement: data missing

25. Conclusions II • Correlations higher after recalculation and refinement, though most of them still weak • Highest correlation: precision vs. Ramachandran score & structural uncertainty (QUEEN)

26. Acknowledgements • Utrecht University Alexandre Bonvin Rob Kaptein • EBI Cambridge Wim Vranken • CESG/BMRB Jurgen Doreleijers Zachary Miller Eldon Ulrich John Markley • Radboud University Nijmegen Chris Spronk Sander Nabuurs • RIKEN Japan Peter Güntert • Institut Pasteur Paris Michael Nilges