Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography.

Ali Ebrahim,Danny Axford,Michael A Hough,Richard W Strange,John Beale,Tadeo Moreno-Chicano,Robin L Owen,Martin V Appleby,Darren A Sherrell

doi:10.1107/s2059798318010240

Ali Ebrahim, Danny Axford + Show 7 more

Open Access

https://doi.org/10.1107/s2059798318010240

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Abstract

The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.

Highlights

X-ray crystallography using synchrotron radiation is at the core of structural biology, providing atomic-level insight into key biological processes
Polymorphism and non-isomorphism of crystals have been a challenge in protein crystallography since the earliest days of the field, with an early example of non-isomorphism being the separation of lysozyme into type I and type II in the 1960s to allow the structure determination of lysozyme
A total of 20 successive images of 20 ms exposure each were measured at each aperture position of a chip, providing 20 room-temperature data sets at different dose points

Summary

Introduction

X-ray crystallography using synchrotron radiation is at the core of structural biology, providing atomic-level insight into key biological processes. In the case of multi-crystal and serial micro-crystallography data-set formation, larger scale differences are often used as the basis for the formation of a final data set through brute-force merging or a more refined approach such as hierarchical cluster analysis (Foadi et al, 2013; Santoni et al, 2017) Statistical approaches such as the use of a genetic algorithm to optimize data-quality metrics such as the R value or hI/(I)i can be used to obtain a single high-quality data set from many crystals (Zander et al, 2016), in the future this could be used to identify and separate non-isomorphous groups

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