“New” methods
By Paul Ellis
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+
“traditional” “quick”
heavy atom concentration mM M
soaking time ≥ hours ≤ minutes
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
Bromide site
Phased anomalous map
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 fast slow
binding weaker stronger
MAD? yes (K) no
Kr K edge
SP18 by Kr MAD
RAW AFTER wARP
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
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-)
Anomalous Patterson
Structure solution
SHARP
DM
wARP
<ΔF±>/<F> ≈ 2.5%
X-ray absorption edges
Accessible energies
Missing edges
Missing elements
Incorrect Correct
Sulfur anomalous
Xenon anomalous
Uranium anomalous
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
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
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