Versatile microporous polymer-based supports for serial macromolecular crystallography.

Isabelle Martiel,Celestino Padeste,Juliane John,Oskar Aurelius,Maximilian Wranik,Chia-Ying Huang,Ching-Ju Tsai,Meitian Wang,John H Beale,Jonas Mühle,May Marsh,Martin Högbom,Laura Vera,Agnieszka Karpik,Natacha Olieric

doi:10.1107/s2059798321007324

Isabelle Martiel, Celestino Padeste + Show 13 more

Open Access

https://doi.org/10.1107/s2059798321007324

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Abstract

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

Highlights

Serial crystallography currently encompasses two datacollection methods for modern structural biologists: singleshot, still exposures on thousands of microcrystals and small-rotation wedges from several to hundreds of microcrystals. The former can be performed at both X-ray freeelectron laser (XFEL) and synchrotron light sources, whereas the latter can only be performed at a synchrotron
At XFELs, the high intensity of the X-ray pulses impedes the collection of multiple diffraction patterns at a given position, making serial femtosecond crystallography (SFX) the only possible data-collection method
Single-shot, still image data-collection methods at synchrotrons were driven by the XFEL SFX methods and spawned serial synchrotron crystallography (SSX; Stellato et al, 2014; Botha et al, 2015; Diederichs & Wang, 2017)

Summary

Introduction

Serial crystallography currently encompasses two datacollection methods for modern structural biologists: singleshot, still exposures on thousands of microcrystals and small-rotation wedges from several to hundreds of microcrystals. The former can be performed at both X-ray freeelectron laser (XFEL) and synchrotron light sources, whereas the latter can only be performed at a synchrotron. Background curves were measured at different places on the chip (Fig. 5a, inset). The basal air scattering was subtracted to obtain the curves corresponding to the individual contributions of the different components of the sample, solution and mount (Fig. 5b)

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