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Efficient Derivatization of Macromolecules Using Quick Soaks for Structural Analysis

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katima
21 diapositives · 336 Views · Oct 04, 2014

The study explores innovative quick soak methods for derivatizing macromolecules, offering a time-efficient alternative to traditional soaking processes. It discusses the impact of high ion concentrations, including bromide and noble gases, on crystal quality and resolution. Notably, quick soaks reveal significant advantages such as better resolution and anomalous peak visibility compared to longer methods. This approach is highlighted for its potential in high-throughput applications, emphasizing recent advancements in software and robotic automation for structural genomics and macromolecular phasing.

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Efficient Derivatization of Macromolecules Using Quick Soaks for Structural Analysis
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  1. “New” methods By Paul Ellis

  2. Derivatizing with quick soaks • Quick soaks can be much less time consuming than traditional long soaks or cocrystallizing • 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 analog for mammalian lysyl oxidase: • 70 kDa glycoprotein • 1 intrinsic Cu • 4 molecules in the asymmetric unit • Tried long soaks and cocrystallizing 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. Krypton & Xenon • Underutilized • More isomorphous than traditional derivatives • Must be stable in cryoprotectant • Good chance of useful derivative • Quillin: large-to-small mutations

  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