“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