3D-printed holders for in meso in situ fixed-target serial X-ray crystallography.

Chia-Ying Huang,Vincent Olieric,Martin Caffrey,Meitian Wang,Nathalie Meier

doi:10.1107/s1600576720002897

Chia-Ying Huang, Vincent Olieric + Show 3 more

Open Access

https://doi.org/10.1107/s1600576720002897

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Abstract

The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient post-crystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

Highlights

In meso or lipid cubic phase crystallization has played an important role in X-ray structure determination of membrane proteins, especially G-protein coupled receptors and the complexes they form (Cherezov et al, 2007; Jaakola et al, 2008; Chien et al, 2010; Wu et al, 2010; Caffrey, 2015)
The new holders are easy to assemble and load with sachets cut from IMISX plates and lend themselves to convenient ligand and heavy-atom soaking and screening of crystals in situ in a protective environment
The holders can be used to store samples under cryogenic conditions for subsequent high-throughput screening with automated sample changers

Summary

Introduction

In meso or lipid cubic phase crystallization has played an important role in X-ray structure determination of membrane proteins, especially G-protein coupled receptors and the complexes they form (Cherezov et al, 2007; Jaakola et al, 2008; Chien et al, 2010; Wu et al, 2010; Caffrey, 2015). A Y-shaped support was introduced subsequently to facilitate data collection over a large flat – as opposed to a curved – sample area This holder proved to be time consuming to assemble and not practical to use (Huang et al, 2018). They are extremely easy to assemble – from two identical pieces They provide increased mechanical stability, they limit ice formation when collecting diffraction data in a cryogenic stream and they facilitate post-crystallization treatments. The second, h2, was designed for samples in which the more fluid variant of the cubic phase, the sponge phase (Cherezov et al, 2006; Caffrey, 2015), has formed Both holders can be used for roomtemperature (RT, 293 K) data collection and are compatible with sample-changing robots.

Design and use of 3D-printed IMISX sample holders

Post-treatment capabilities of the 3D-printed IMISX sample holders

Data collection with the 3D-printed IMISX sample holders

Discussion and remarks

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