1. “New” methods By Paul Ellis

2. Derivatizing with quick soaks • Quick soaks can be much less time consuming than traditional long soaks or cocrystallising • High concentrations can be destructive of crystal order • Ions used include: • Br-, I- • Cs+, Rb+ • Gd3+, Ho3+, Sm3+, Eu3+

3. PPLO • Pichia pastoris lysyl oxidase – an analogue for mammalian lysyl oxidase: • 70 kDa glycoprotein • 1 intrinsic Cu • 4 molecules in the asymmetric unit • Tried long soaks and cocrystallising with: • Hg(II), Yb(III), Sm(III), PIP, EMTS, WO42-, IrCl42-, Os(III), Kr… • Poor resolution • No peaks in anomalous Patterson • Tried short soaks in KBr • ≥ 1.0 M, ≥ 120 s destroyed crystal • Good resolution • Good peaks in anomalous Patterson with 90 s, 0.75 M

4. Bromide site

5. Phased anomalous map

6. Experimental Experimental Energies for MAD were chosen using a fluorescence scan from each crystal Energies for MAD were chosen using a fluorescence scan from each crystal Mb rotation angle /image: 0.5° exposure time: 15 s total rotation range: 180° resolution range: 17-1.7 Å measured reflections: 230000 unique reflections: 23000 completeness: 99.6% anom. completeness: 99.6% multiplicity: 10.0 I/(I) (overall): 26.6 I/(I) (1.71-1.70 Å): 11.4 Rmerge: 0.065 Mb rotation angle /image: 0.5° exposure time: 15 s total rotation range: 180° resolution range: 17-1.7 Å measured reflections: 230000 unique reflections: 23000 completeness: 99.6% anom. completeness: 99.6% multiplicity: 10.0 I/(I) (overall): 26.6 I/(I) (1.71-1.70 Å): 11.4 Rmerge: 0.065 SP18 rotation angle /image: 0.5° exposure time: 15 s total rotation range: 180° resolution range: 24-2.0 Å measured reflections: 730000 unique reflections: 18800 completeness: 99.6% anom. completeness: 99.9% multiplicity: 38.0 I/(I) (overall): 30.0 I/(I) (2.02-2.00 Å): 1.41 Rmerge: 0.097 SP18 rotation angle /image: 0.5° exposure time: 15 s total rotation range: 180° resolution range: 24-2.0 Å measured reflections: 730000 unique reflections: 18800 completeness: 99.6% anom. completeness: 99.9% multiplicity: 38.0 I/(I) (overall): 30.0 I/(I) (2.02-2.00 Å): 1.41 Rmerge: 0.097 Krypton & Xenon • Underutilized • More isomorphous than traditional derivatives • Must be stable in cryoprotectant • Good chance of useful derivative • Quillin: large-to-small mutation to create binding site

7. Kr K edge

8. SP18 by Kr MAD RAW AFTER wARP

9. SAD v. MAD • SAD will be the method of choice for high throughput • Programs designed for SAD data are becoming available • Robots will give experimenters more freedom to try SAD

10. HIBADH – a SAD example • 3-hydroxyisobutyrate dehydrogenase • a ubiquitous enzyme involved in valine catabolism • 1 Crystal • P43212, 103 × 103 × 108 Å • 2 × 295 residues in asu ≈ 70 kDa • Grown in 5 mM Pb2+ • Data collection • Δφ = 94° • dmin = 2.2 Å • R = 9.7% • Multiplicity = 7.6 • <I/σ(I)> = 15 • λ = 0.79 Å (Pb f " ≈ 10e-)

11. Anomalous Patterson

12. Structure solution SHARP DM wARP <ΔF±>/<F> ≈ 2.5%

13. X-ray absorption edges

14. Accessible energies

15. Missing edges

16. Missing elements Incorrect Correct

17. Sulfur anomalous

18. Xenon anomalous

19. Uranium anomalous

20. Summary • Quick soaks • Dauter, Z., Li., M., & Wlodawer, A. (2001). Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics. Acta Cryst. D57, 239-249. • Nagem, R.A.P., Dauter, Z., & Polikarpov, I. (2001). Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers. Acta Cryst. D57, 996-1002. • Kr and Xe • Cohen, A.E., Ellis, P.J., Kresge, N. & Soltis, S.M. (2001). MAD phasing with krypton. Acta Cryst. D57, 233-238. • Quillin, M.L., & Matthews, B.W. (2002). Generation of noble-gas binding sites for crystallographic phasing using site-directed mutagenesis. Acta Cryst. D58, 97-103. • SAD • Dauter, Z., Dauter, M., & Dodson, E. (2002). Jolly SAD. Acta Cryst. D58, 494-506. • Low energy

21. Acknowledgements • School of Molecular & Microbial Biosciences, University of Sydney: • Hans Freeman • Mitchell Guss • Anthony Duff • Department of Chemistry & Biochemistry, Montana State University: • David M. Dooley • Department of Molecular Biochemistry, Ohio State University: • Russ Hille • Thomas Conrads • Structural Molecular Biology, SSRL: • Peter Kuhn • Mike Soltis • Aina Cohen • Nancy Fathali • Department of Energy: • Office of Basic Energy Sciences • Office of Biological and Environmental Research • National Institutes of Health, National Center for Research Resources, Biomedical Technology Program