1 / 21

Innovative Crystallography Methods for Derivatizing Protein Crystals

Explore new methods like quick soaks and derivatizing with ions for protein crystallography. Discover the benefits of using techniques like MAD and SAD for structure determination.

kalei
Télécharger la présentation

Innovative Crystallography Methods for Derivatizing Protein Crystals

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related