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.
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.
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).
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 types (e.g. mammalian cells), we describe here the procedures for insect cells. The protocol assumes that over-expression of the protein of interest has already been achieved. Methods for the production of a recombinant baculovirus and its use for expression of the protein of interest are available in references 23,24,25,26,27.
1. Identification of Crystal-containing Cells
2. Sample Purification
Note: We describe two methods of analysis of the in vivo crystals from purified crystals and in cellulo, respectively.
Figure 3: Purified In Vivo Microcrystals. Representative purification of crystals of the BmCPV1 polyhedrin from Sf9 insect cells showing (a) pelleted cell debris and crystals after sonication, and (b) close-up on the debris and crystal layers. Debris layer can be carefully resuspended in the supernatant and discarded for faster crystal purification. (c) Optical microscopy image of purified polyhedra. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: Cell-sorting. Representative flow cytometry scatter plots from (a) non-infected cells (mock), which do not contain crystals, and (b) Sf9 cells infected by a recombinant baculovirus over-expressing for the BmCPV1 polyhedrin. Differences in the scattering pattern between the two populations allowed gating of the crystal-containing cell population (red gate, high SSC values). Each plot represents 20,000 sorting events. SSC: side light scattering; FSC: forward light scattering. (c) Optical microscopy image of a representative population of the sorted cells, showing the presence of intracellular crystals. Scale = 50 µm. Please click here to view a larger version of this figure.
3. Sample Preparation for Data Collection
Note: If cells or crystals are to be derivatized for experimental phasing follow protocol detailed in section 3.1. Otherwise, go directly to sections 3.2 for purified crystals or 3.3 for in cellulo analysis.
4. Data Collection
Note: Parameters used for data collection are given as a guide and should be optimized for each crystal type and synchrotron beamline.
Figure 5: Data Collection Strategy from Samples on Micromesh Support. (a) Strategy of data collection per lane; alignment is done at the beginning of each lane (dots) and data is collected along the lane. (b) Close-up of a trypan blue-stained, crystal-containing cell on a mesh, as seen on the microcrystallography beamline screen. (c) Close-up of a purified polyhedra on a mesh, as seen on the microcrystallography beamline screen. The mesh squares are 25 µm x 25 µm. Please click here to view a larger version of this figure.
5. Data Processing
NOTE: Here, we only mention briefly the steps of data processing that are specific to serial microcrystallography, where data from multiple crystals are merged. Many excellent articles are available describing data processing in depth31,32,33,34,35,36 and structure determination is beyond the scope of this protocol.
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 until a sufficient level of purity is reached as judged from the white and chalky aspect of the pellet (Figure 3c).
For the in cellulo approach, flow cytometry profile of non-infected cells is compared with profile of crystal-containing cells and used to determine which cell population should be selected to sort crystal-containing cells away from the other cells (Figure 4). In this example, cells containing polyhedra have a higher side-scattering than non-infected cells. Other differences in scattering patterns may be observed for other crystal types and gating should be modified accordingly.
Purified microcrystals or crystal-containing cells are pipetted onto a micromesh (Figure 5). Cells stained with trypan blue can be easily visualized using cameras available at most of synchrotron beamlines (Figure 5b), while purified crystals are laborious to identify and align with the X-ray beam due to their small size (Figure 5c). When collecting data, it is best to proceed in a grid pattern in order to minimize the centering procedures, and to avoid exposing twice the same cell or crystal, or missing any (Figure 5a). Data collected should be processed carefully to account for differences in diffraction limits, the effect of radiation damage and a possible lack of isomorphism.
Figure 1: Overview of Two Methods for Structure Determination using In Vivo Microcrystals. Microcrystals are produced in cell culture by over-expression of the protein of interest using a recombinant expression system. In specific cases, microcrystals may also be produced naturally by the cells in particular conditions. The top row shows the classical approach where cells are lysed and crystals are purified by differential centrifugation. Purified microcrystals are pipetted onto a micromesh. After removal of the excess liquid, a cryoprotectant solution is added, excess liquid is blotted again and the mesh is flash-cooled. For X-ray diffraction experiments, crystals are carefully aligned with the X-ray beam, which may be the rate limiting step in data collection. The bottom row presents the in cellulo approach. Cells are sorted by flow-cytometry to specifically select crystal-containing cells. Cells are stained with trypan blue to facilitate visualization and spread onto a micromesh. In this case, the addition of a cryoprotectant is optional. For X-ray diffraction experiments, cells are easily visualized using typical cameras available at synchrotron beamlines facilitating the centering process.
Image of automated liquid chromatography system courtesy of GE Healthcare AB, Uppsala, Sweden. Please click here to view a larger version of this figure.
Figure 2: Examples of In Vivo Microcrystals. Selected examples of in vivo crystals: (a) Sf9 cells with cypovirus polyhedra; (b) LD652 cells with entomopoxvirus spheroids; (c) Bacillus thuringiensis with Cry3A crystals (arrow); (d) Sf9 cells with Trypanosoma brucei cathepsin B crystals; (e) Sf21 cells with calcineurin crystals (arrows); (f) COS7 cells monolayer with PKA:Inka1 crystals; (g) CHO cells with IgG crystals. Reproduced from14,18,29,37,39,42, with permission. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Table 1: Summary of In Vivo Crystals Grown in Cultured Cells Expressing Recombinant Protein. Please click here to view a larger version of this figure.
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-like crystals grown in insect cells are rigid and highly resistant to mechanical stress, and could be purified using a protocol similar to the one described here 14. On the other hand, firefly luciferase crystals, also grown in cultured insect cells and with a similar needle-like morphology, immediately dissolve upon cell lysis 38. Thus, the protocol for extraction and purification of in vivo crystals will need to be adapted in a trial-error basis for particular cases as described in steps 1.3, 2a.3-4, 2b.4, 3.1.3-4 and 3.2a.5.
For fragile crystals, (partial) separation from cell debris might be achieved by low speed centrifugation (i.e. 200 x g) or addition of a sucrose cushion at the bottom of the tube instead of repeated sonication. As a general rule, crystal samples prepared for diffraction data collection purposes do no need to be highly purified. Thus, rapid purification is preferable to cryo-cool the crystals before they might decay.
Application to in vitro and in vivo microcrystals
Although both protocols are here illustrated with the analysis of in vivo grown crystals, the methodology can be readily used for microcrystals grown in crystal trays from purified recombinant protein. In this case, the following modifications should be considered: i) the mesh support may be used to harvest the microcrystals directly from the crystallization drop, rather than transferring them by pipetting; ii) since the amount of crystals may be a limiting factor, special care will need to be taken when blotting the excess of liquid from the mesh support, to avoid excessive removal of crystals; iii) different cryoprotectant buffers will need to be tested, as done in conventional crystallography (see reference 30).
On the other hand, for in vivo-grown crystals, the in cellulo approach is strongly recommended. Because cells are easier to visualize and manipulate than purified crystals, this approach is more efficient in terms of beamtime usage and more accessible to researchers with little or no experience in microcrystallography. It is also more suitable for fragile crystals that may be affected by the extraction and purification from the cell due to changes in the chemical environment, dilution of the surrounding proteins and mechanical damage. Besides, cells provide a protective environment to the crystals, alleviating the need for the addition of a cryoprotectant. Omission of the cryoprotection treatment has been previously shown to prevent accumulation of defects and improved the isomorphism between individual crystals, for data collection at room temperature 40. The in cellulo protocol also bypasses the requirement for incubation in cryoprotectant solution, while still allowing data collection at cryogenic temperature. Overall, in cellulo data collection has two main advantages: 1) it may produce data of better quality and higher resolution for a given period of beamtime 20; and 2) it maintains the crystallized protein in a cellular environment increasing the chances of retaining a biologically-relevant ligand.
Limitations of the technique
An intrinsic complication of serial microcrystallography is the important number of partial datasets generated during data collection. Consequently, data processing becomes an increasingly time-consuming process. However, the basic workflow for data processing can largely be automated. For example, the optimal selection of isomorphous crystals can be determined by hierarchical cluster analysis28, for instance implemented in BLEND36. In addition, programs developed for the analysis of X-ray free electron lasers (XFEL) can also be adapted to process serial microcrystallography data collected on synchrotron beamlines2 and, at some beamlines automated rastering of mesh and crystal detection have been implemented greatly facilitating serial data collection2,41.
The authors have nothing to disclose.
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.
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. |