“New” methods By Paul Ellis

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+

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 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

<ΔF±>/<F> ≈ 2.5% 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