Microcrystallography of Protein Crystals and In Cellulo Diffraction
Summary
A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.
Abstract
The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 µm in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.
Introduction
The use of X-ray crystallography for the determination of high-resolution structures of biological macromolecules has experienced a steady progression over the last two decades. The growing uptake of X-ray crystallography by non-expert researchers exemplifies the democratization of this approach in many fields of life sciences1.
Historically, crystals with dimensions below ~10 µm have been considered as challenging, if not unusable, for structure determination. The increasing availability of dedicated microfocus beamlinesat synchrotron radiation sources worldwide and technological advances, such as the development of tools to manipulate microcrystals, have removed much of these barriers that stymied the wide use of X-ray microcrystallography. Advances in serial X-ray microcrystallography2,3 and micro electron diffraction4 have shown that the use of micro- and nanocrystals for structure determination is not only feasible but also sometimes preferable to the use of large crystals5,6,7.
These advances were first applied to the study of peptides8 and natural crystals produced by insect viruses9,10. They are now used for a diverse range of biological macromolecules including the most difficult systems such as membrane proteins and large complexes11. To facilitate the analysis of these microcrystals, they have been analyzed in meso, particularly membrane proteins12 and in microfluidics chips13.
The availability of these novel microcrystallography methodologies has raised the possibility of using in vivo crystallization as a new route for structural biology14,15,16 offering an alternative to classical in vitro crystallogenesis. Unfortunately, even when in vivo crystals can be produced, several obstacles remain such as the degradation or loss of ligands during the purification from cells, difficulty in the manipulation and visualization of the crystals at the synchrotron beamline and tedious X-ray diffraction experiments. As an alternative crystals have also been analyzed directly within the cell without any purification step17,18,19. A comparative analysis suggests that such in cellulo approaches may be more efficient than the analysis of purified crystals and yield data of higher resolution20.
This protocol is intended to assist researchers new to protein microcrystallography. It provides methodologies focusing on sample preparation and manipulation for X-ray diffraction experiments at a synchrotron beamline. Two options are proposed using isolated crystals for classical microcrystallography or crystal-containing cells sorted by flow cytometry for in cellulo analysis (Figure 1).
Protocol
Representative Results
Discussion
This protocol provides two approaches to analyze microcrystals with the aim of facilitating the analysis of very small crystals that would have been overlooked in the past.
Critical steps for microcrystal purification
The presented protocol has been optimized using Bombyx mori CPV1 polyhedrin expressed in Sf9 cells as a model system. However, in vivo microcrystals display a great variability in mechanical resistance. For instance, cathepsin B needle-lik…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Chan-Sien Lay for providing pictures of purified microcrystals, Daniel Eriksson and Tom Caradoc-Davies for support at the MX2 beamline of the Australian Synchrotron, and Kathryn Flanagan and Andrew Fryga from the FlowCore facility at Monash University for their invaluable assistance.
Materials
| Sf9 cells | Life Technologies | ||
| SF900-SFM insect medium | Life Technologies | ||
| 1L cell culture flask | Thermofisher Scientific | ||
| Shaking incubator for insect cell culture | Eppendorf | ||
| 50mL conical tubes | Falcon | ||
| Centrifuge with swing buckets for 50mL tubes | Eppendorf | ||
| Sonicator equiped with a 19mm probe | MSE Soniprep 150 | ||
| Glass slides | Hampton Research | ||
| Hemacytometer | Sigma-Aldrich | ||
| Propidium iodide | Thermofisher Scientific | ||
| BD Influx cell sorter | BD Biosciences | ||
| Hampton Heavy atom screens | Hampton Research | ||
| Microcentrifuge | Eppendorf | ||
| Micromesh | Mitigen | 700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies. | |
| Paper wick | Mitigen | The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used. | |
| Ethylene glycol | Sigma-Aldrich | ||
| Trypan blue | Life Technologies | ||
| MX2 microfocus beamline | Australian Synchrotron | A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein Microcrystallography. Australian Journal of Chemistry 67 (12), 1793–1806, doi:10.1071/CH14455. |
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