1. Don't fffear the buccaneer Kevin Cowtan, York. ● Map simulation ⇨ A tool for building robust statistical methods ● 'Pirate' ⇨ A new statistical phase improvement method ● 'Buccaneer' ⇨ A new statistical chain tracing method ● Results ⇨ And a diatribe about their irrelevance The Royal Society York Structural Biology Laboratory

2. Map simulation The Royal Society York Structural Biology Laboratory Refined model density. Target noisy map. Simulated noisy map. Structure factors Known (reference) structureUnknown (work) structure Phases Scale factors Phase errors Map simulation is a tool to generate problem specific statistical targets:

3. Map simulation: Method The Royal Society York Structural Biology Laboratory Low |E| Med. |E| High |E| Med. resol. High resol. Low resol. Low |E| Med. |E| High |E| Med. resol. High resol. Low resol. Transferring the errors: 1. Classify the reflections from both structures by |E| and resol. (Note: we use 225 bins, not 9!)

4. Map simulation: Method The Royal Society York Structural Biology Laboratory Low |E| Med. |E| High |E| Med. resol. High resol. Low resol. Low |E| Med. |E| High |E| Med. resol. High resol. Low resol. 0.1, 0.0 0.0, 0.0... 0.9, 0.8 0.6, 0.4... Transferring the errors: 2. Copy FOMs by bin from work structure to reference. (We pick a random FOM from the same bin of the work structure for each reflection in the reference structure.)

5. Map simulation: Method The Royal Society York Structural Biology Laboratory P(  )  0   Transferring the errors: 3. Simulate a phase error in accordance with the distribution for that FOM:

6. Map simulation: Method The Royal Society York Structural Biology Laboratory |E| 2 Resolution |E| 2 Resolution Transferring the scales: Rescale the reference data to match the work data, after accounting for the difference in cell volumes.

7. Map simulation: Method The Royal Society York Structural Biology Laboratory Result: Map calculated from simulated reference data has same statistical properties as work map. Notes: Need reliable FOMs! Can potentially simulate HL coeffs too. Should bin FOMs for centric/acentric data separately (if data available).

8. 'Pirate': Rationale Density modification history has been dominated by the solvent mask in one form or another. Limitations: –What do we do with disordered protein? –What do we do with ordered solvent? –Need to know solvent content. –What do we do for non-proteins? The Royal Society York Structural Biology Laboratory

9. 'Pirate': Method Divide map into a multi-dimensional continuum of states. The Royal Society York Structural Biology Laboratory e.g. Local mean and local variance classify map into: ● Electron sparse/dense ● Disordered/ordered Dense, ordered Dense, disordered Sparse, ordered Sparse, disordered

10. 'Pirate': Method Compare simulated and known map to obtain density distributions for each region, then apply these distributions to the unknown map. The Royal Society York Structural Biology Laboratory Reference structure:Work structure:

11. 'Pirate': Method Obtain per-grid density probability distributions –Also allows NCS, known density etc. Transform using equations of Bricogne (1992). –Similar to Terwilliger (1999). –Map probability becomes phase probability distribution. The Royal Society York Structural Biology Laboratory Bricogne (1992) Proc. CCP4 Study Weekend Bricogne (1997) Methods in Enzymology R I

12. 'Pirate': Method The Royal Society York Structural Biology Laboratory Finally, combine new distribution with original HL coefficients, for new phases and maps. Gives final 'improved' phase probabilities. R I R I X ABCD

13. 'Pirate': Method The Royal Society York Structural Biology Laboratory Notes: No solvent content required, since reference map is pre-scaled to work map. Single step process (for now) –No solvent mask -> no mask to refine. Should work for novel problems too (with related reference structure) –e.g. No solvent, disordered domains, metaloproteins.

14. 'Buccaneer': Method Compare simulated map and known model to obtain likelihood target, then search for this target in the unknown map. The Royal Society York Structural Biology Laboratory Reference structure:Work structure: LLK

15. 'Buccaneer': Method Compile statistics for reference map in 4A sphere about C  => LLK target. The Royal Society York Structural Biology Laboratory 4A sphere about Ca also used by 'CAPRA' Ioeger et al. (but different target function). Use mean/variance (in future histogram).

16. 'Buccaneer': Method Find candidate C  positions using LLK-fffear search. (~1 per 3 residues) The Royal Society York Structural Biology Laboratory

17. 'Buccaneer': Method Extend from candidates using 2 residue lookahead with Ramachandran restraints. (Same target-fn. but in real space) Then ARP/wARP? The Royal Society York Structural Biology Laboratory Lookahead search c.f. Jones, Oldfield, Terwilliger, etc.

18. Results Problem: “tuning” of one program to another. The Royal Society York Structural Biology Laboratory E corr / MPE w / m 0 Phasing Ph.Impr. E corr / MPE w / m 0 'dm' 'resolve' 'dm' 'resolve' 'mlphare' 'solve' 0.508 / 59.1 / 1.35 0.474 / 61.0 / 0.83 0.700 / 50.6 / 0.61 0.436 / 67.8 / 0.37 0.750 / 47.7 / 0.68 0.710 / 48.0 / 0.67 'resolve' version 2.0.5, with 'no build' option in order to compare model-free phasing. Statistics are:E corr : E-map correlation; MPE w : weighted Mean Phase Error; m 0 : gradient of regression of cos(  ) vs. FOM What other examples of “tuning” are present in this case?

19. Results After 'solve', but with other tuning problems: The Royal Society York Structural Biology Laboratory E corr / MPE w / m 0 Ph.Impr. 'pirate' 1 'resolve' 'dm'0.750 / 47.7 / 0.68 0.710 / 48.0 / 0.67 0.775 / 43.2 / 1.08 'pirate' 2 'pirate' 3 'pirate' 6 'pirate' 5 'pirate' 4 0.762 / 43.3 / 0.98 0.824 / 37.2 / 1.02 0.788 / 39.7 / 0.94 0.745 / 44.7 / 1.02 0.759 / 42.7 / 0.94 Reference structures Beta-mannosidase (2003) Structure Boraston, Revett, Boraston, Nurizzo, Davies

20. Results The Royal Society York Structural Biology Laboratory SAD'dm'

21. Results The Royal Society York Structural Biology Laboratory 'resolve''pirate'

22. Results Other cases: –MIRAS: Commercial structure phased with MLPHARE. Results better than 'dm'. –High resolution: RNAse phase extension to 1.5, 1.0A. Map improved (unlike 'dm') with appropriate reference structure. (But not as good a dual space methods: ACORN). The Royal Society York Structural Biology Laboratory

23. Future 'Pirate' available soon Q1 2004 (after tuning) 'Pirate' flexi-domain averaging Q3 2004 'Buccaneer' 2004? Technology: Both applications are extremely simple, built using Clipper libraries, less than 1000 lines of code each, less than 2 months development. The Royal Society York Structural Biology Laboratory

24. Conclusions Very simple but effective applications can be built with improved statistical targets from map simulation calculations. Preliminary results on real data suggest this approach is competitive with the state-of-the- art, even at an early stage of development. Need reliable phase probability distributions (figures of merit). The Royal Society York Structural Biology Laboratory

25. Acknowledgements ● G. Bricogne (Original probability transformation eqns.) ● T. Terwilliger (First implementation of statistical dm.) ● E. Dodson (Test data) ● Royal Society (KDC funding)