1. “New” methods
By Paul Ellis

2. Derivatizing with quick soaks
“traditional”
“quick”
heavy atom concentration mM M
soaking time
≥ hours
≤ minutes
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 Kr Xe
“on” rate
fast
slow
“off” rate
binding
weaker
stronger
MAD?
yes (K) no

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
SAD
MAD
number of wavelengths 1
>1
anomalous signal
≥ 2e-/10 kDa
≥ 1e-/10 kDa
high redundancy?
yes no

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. <ΔF±>/<F> ≈ 2.5%
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,
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,
Kr and Xe
Cohen, A.E., Ellis, P.J., Kresge, N. & Soltis, S.M. (2001). MAD phasing with krypton. Acta Cryst. D57,
Quillin, M.L., & Matthews, B.W. (2002). Generation of noble-gas binding sites for crystallographic phasing using site-directed mutagenesis. Acta Cryst. D58,
SAD
Dauter, Z., Dauter, M., & Dodson, E. (2002). Jolly SAD. Acta Cryst. D58,
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