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 developme....

Protocol

Note: In vivo crystallization has been reported in many organisms including in bacteria, yeast, plants, insects and mammals (reviewed in reference ²¹). Crystallization of recombinant proteins has also been achieved in the laboratory using transient transfection of mammalian cells and baculovirus infection of insect cells. The following protocol has been developed using the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin gene cloned in a recombinant baculovirus under the baculovirus polyhedrin promoter, generated as per instructions in reference ²². Thus, although this protocol may be adapted to other cell t....

Results

An overview of both alternative methods for structure determination using in vivo microcrystals is presented (Figure 1). Polyhedra can easily be purified by sonication and centrifugation. Due to their density, they form a layer at the bottom of the tube underneath a layer of debris that can be removed by pipetting (Figure 3a and 3b). The sample is then subjected to several rounds of sonication and washes.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sf9 cells	Life Technologies
SF900-SFM insect medium	Life Technologies
1 L cell culture flask	Thermofisher Scientific
Shaking incubator for insect cell culture	Eppendorf
50 mL conical tubes	Falcon
Centrifuge with swing buckets for 50 mL tubes	Eppendorf
Sonicator equiped with a 19 mm 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.