Low-dose in situ prelocation of protein microcrystals by 2D X-ray phase-contrast imaging for serial crystallography.

Isabelle Martiel,Bill Pedrini,Meitian Wang,Marco Stampanoni,Shibom Basu,Chia-Ying Huang,Martin Caffrey,Oliver Bunk,Ezequiel Panepucci,Pablo Villanueva-Perez

doi:10.1107/s2052252520013238

Abstract

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

Highlights

Macromolecular crystallography (MX) data collection at X-ray free-electron lasers (XFELs) requires the merging of still images from many crystals exposed to intense femtosecond X-ray pulses, its commonly given name of serial femtosecond crystallography (SFX) (Chapman et al, 2011; White et al, 2012, 2016)
In the X-ray images, crystals appear as objects of moderate contrast, resulting from the edge-enhancing phase contrast of free-space propagation of the X-ray beam after the sample (Paganin et al, 2002)
A residual phase-contrast halo was still observed around the metal beads owing to the semi-empirical tuning of the phase-retrieval step being targeted on the crystals (Fig. S4), but this does not interfere with their automatic selection thanks to the sufficient contrast

Summary

Introduction

Macromolecular crystallography (MX) data collection at X-ray free-electron lasers (XFELs) requires the merging of still images from many crystals exposed to intense femtosecond X-ray pulses, its commonly given name of serial femtosecond crystallography (SFX) (Chapman et al, 2011; White et al, 2012, 2016). Among the fixed-target approaches, the double-sandwich in situ method (Axford et al, 2016; Broecker et al, 2016; Huang et al, 2015, 2016, 2018) was used both at room temperature or in cryogenic conditions This method is characterized by a flat geometry, since the sample was grown between two thin and flat films. The double-sandwich in situ method is of particular relevance for small dose-sensitive crystals, which are hardly amenable to harvesting and single-crystal data collection, e.g. membrane-protein crystals Serial methods make it possible to expose these small crystals to their maximum safely tolerable dose over a small rotation range, thereby maximizing resolution, completeness and multiplicity by merging data from a number of crystals

Methods

Results

Conclusion