A crystal-processing machine using a deep-ultraviolet laser: application to long-wavelength native SAD experiments.

Masahide Hikita,Toshiya Senda,Naohiro Matsugaki,Masaki Yamamoto,Yoshiaki Kawano

doi:10.1107/s2053230x2101339x

Abstract

While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed. The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.

Highlights

X-ray crystallography is one of the primary methods used for the three-dimensional structure determination of proteins at atomic resolution
We have developed a crystal-processing machine using a deep-UV laser to improve diffraction data quality with longwavelength X-rays
We observed a significant improvement in diffraction data quality, probably due to a substantial reduction of background noise and errors from noncrystalline

Summary

Introduction

X-ray crystallography is one of the primary methods used for the three-dimensional structure determination of proteins at atomic resolution. A specific strategy is needed to obtain high-quality diffraction data because the longwavelength X-rays used in the native SAD method are affected by the solvent, the cryoloop and the size and shape of the crystal (Liebschner et al, 2016; Basu et al, 2019). To reduce the errors originating from a mounted crystal, it is necessary to remove the unnecessary portions of the crystal and process the remainder into the desired shape. For these purposes, a laser-processing technique would be ideal. Crystal processing using PULSA was originally developed to extract a good portion of a sample crystal, it is quite useful for removing unnecessary parts of the crystal for long-wavelength X-ray experiments. We report a crystal-processing machine using a deep-UV laser for efficient sample fabrication and demonstrate native SAD experiments as a successful application

Laser source

Laser optics

Sample holding and manipulation

Control software

Applications in protein crystals

Native SAD experiments

Methods

Findings

Conclusions and future prospects