Long-wavelength native-SAD phasing: opportunities and challenges.

Shibom Basu,Yusuke Yamada,Yoshiaki Kawano,Meitian Wang,Oliver Bunk,Natacha Olieric,Kay Diederichs,Chia-Ying Huang,Filip Leonarski,Tomizaki Takashi,Vincent Olieric,Justyna A Wojdyla,Masaki Yamamoto,Naohiro Matsugaki,Jerome Basquin,Laura Vera

doi:10.1107/s2052252519002756

Abstract

Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 Å is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.

Highlights

Most of our knowledge about the 3D atomic structure of biological macromolecules is derived directly or indirectly from experimental phasing (EP) in macromolecular crystallography (MX) thanks to the large success of selenomethionine incorporation and heavy-atom derivatization (Hendrickson, 2014)
To study the optimal wavelength for native-single-wavelength anomalous dispersion (SAD) phasing, the measured anomalous signal per absorbed X-ray dose needs to be compared at different wavelengths
Optimization of native-SAD phasing experiments at wavelengths >2 Ais being addressed at dedicated MX beamlines with reduced air absorption and scattering effects, as well as special detector geometry

Summary

Introduction

Most of our knowledge about the 3D atomic structure of biological macromolecules is derived directly or indirectly from experimental phasing (EP) in macromolecular crystallography (MX) thanks to the large success of selenomethionine incorporation and heavy-atom derivatization (Hendrickson, 2014). The sized ‘naked’ crystal, i.e. one without any surrounding solvent relatively low flux and large beam size of such sources have or a loop, in an ideal experiment [Fig. 1(a)]. In limited their application to large and well diffracting crystals. In limited their application to large and well diffracting crystals. addition to the crystal itself, any material in the X-ray beam-

Methods

Results

Conclusion