Protein crystallization occurs as a native process in vivo
. Prominent examples of this biological self-assembly include storage proteins in seeds, enzymes within peroxisomes and insulin within secretory granules1
. Cells seem to control interactions of these proteins through changes in the ionic environment, proteolysis of precursor proteins or specific binding partners. Although these structures regulate cellular functions, in vivo
crystallization has been perceived as atypical behavior and has therefore been neglected in comparison to the considerable efforts devoted to understanding and optimizing in vitro
protein crystallization for X-ray structure determination.
Heterologously expressed proteins are also able to form crystals within cells. In the baculovirus-Sf9 expression system, virions are coated with a crystalline polyhedrin matrix2
, representing a functional biological crystallization system. Site-specific transposition of an expression cassette into a baculovirus shuttle vector replaces the polyhedrin gene with a gene of interest. The permanent activation of the polyhedrin gene promoter ensures high local protein concentrations, obviously one prerequisite for crystal formation in vivo
. This system has been used in insect cells to successfully crystallize polyhedrin3
and chimeric proteins consisting of a polyhedrin part attached to the protein of interest4
, as well as two polyhedrin-free subunits of calcineurin5
. However, no structural data from in vivo
–grown, polyhedrin-free crystals have been obtained so far, a gap largely attributed to the small size of the crystals, limited by the maximum diameter of the living cell.
In this study we present an approach for structural biology that combines the recently established method of serial femtosecond X-ray crystallography (SFX)6
and the use of in vivo
–grown crystals to record diffraction data. The SFX method uses coherent X-ray pulses produced by an X-ray free-electron laser (FEL) that are over a billion times more brilliant than third-generation synchrotron sources and aims to overcome resolution limits imposed by radiation damage at conventional sources7
. Using the ‘diffraction before destruction’ principle, diffraction patterns are collected with single, ultrafast pulses that essentially terminate before the onset of significant structural changes occurs, and the X-ray pulse finally vaporizes the sample6
. The single-pulse diffraction pattern has been predicted to represent the undamaged crystal injected across the beam in a liquid microjet, depending on the pulse intensity and duration8
. As a first proof of principle, a recent study has recorded tens of thousands of snapshots from individual crystals of photosystem I (PSI) at room temperature (17–20 °C), ranging in size from 200 nm to 2 µm (ref. 6
). The successful structural investigation of this protein complex up to a resolution of 8.5 Å demonstrates the viability of using nanocrystals and the SFX method for structure determination.
We observed in vivo
crystallization of polyhedrin-free, glycosylated cathepsin from Trypanosoma brucei
(TbCatB) within Sf9 insect cells transfected with bmon14272 bacmid (Invitrogen) created by on site–specific transposition with pFastBac1 expression plasmid (Invitrogen) containing the Autographa californica
multiple nuclear polyhedrosis virus polyhedrin (PH) promoter. Knockdown of TbCatB has been shown to be lethal for the parasite, which causes human African trypanosomiasis, one of the most important neglected diseases, affecting over 60 million people in central Africa9
. Efficient and cost-effective drugs are not yet available, but cysteine proteases such as TbCatB have been identified as potential drug targets in protozoan parasites10
Approximately 70 h after infection, the formation of needle-shaped microstructures was visible by light microscopy in Sf9 cells infected with recombinant baculovirus containing the gene encoding the pre-pro form of TbCatB (including the TbCatB signal sequence, the pro-peptide and the mature enzyme sequence of TbCatB; ). Electron microscopy (EM) revealed a damaged cell surface and sharp, needle-like crystals 10–15 µm in length and 0.5–1 µm in width spiking out of the cells (). Capsids were visible within the nucleus, and crystals appeared as defined dark areas with sharp square edges within the cytoplasm (). Membranes decorated with ribosomes surrounding the crystals indicate an origin of crystal formation within the endoplasmic reticulum (). An ordered lattice structure observed at higher magnification identified these particles as protein crystals ().
Figure 1 Light microscopic and EM analysis of Sf9 insect cells with embedded in vivo crystals. (a) Light micrograph of Sf9 cells infected with TbCatB virus 90 h after infection. (b) Transmission EM (TEM) micrograph of an embedded and sectioned infected Sf9 cell (more ...)
Usually, protein accumulation is inhibited by the ‘unfolded protein response’ (UPR), in which atypical or misfolded proteins inhibit further protein biosynthesis11
. This transcriptional regulation fails in the case of the polyhedrin promoter, leading to an enormous increase of TbCatB concentrations that finally provokes crystallization. This interpretation is consistent with the observation that changing the signal sequence from the trypanosomal to the insect cell signal peptide led to secretion of soluble TbCatB without crystal formation (data not shown). In contrast to polyhedrin3
, all crystals appeared without embedded virions. During the progress of infection, the number of crystals continuously increased, until more than 70% of the cells contained one or more crystals. If released during infection-mediated lysis, crystals either remained attached to cell remnants or floated freely within the medium.
To isolate in vivo
crystals, we lysed cells and subjected them to differential centrifugation, resulting in ~5 × 105
purified crystals from 106
cells obtained after 8 d in suspension culture. These crystals were stable in deionized water, high- or low-salt solutions and alkaline buffers but became soluble in buffers at pH values below 4. Analysis of the solubilized protein confirmed that the major constituent of the crystals was glycosylated TbCatB comprising the C-terminal residues 63 to 93 of the propeptide and the mature enzyme sequence (residues 94 to 336; Supplementary Note and Supplementary Fig. 1
Synchrotron radiation–based experiments showed that the isolated TbCatB in vivo
–grown crystals were too small to generate suitable X-ray diffraction at beamline X13 of DORIS III (HASYLAB/DESY, Hamburg, Germany), probably owing to technical limitations of the beamline associated with the crystal size rather than to the diffraction quality of the crystal. However, strong diffraction was observed at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). Purified in vivo
crystals were crushed by vigorous stirring with glass beads to increase the particle density and injected across the FEL beam in a vacuum in a stream of water using a liquid jet (Supplementary Fig. 2
). Single-pulse diffraction patterns were recorded at up to 7.5-Å resolution, corresponding to the technical resolution limit determined by the photon energy of the available X-ray pulses (2.0 keV, λ = 6.2 Å) and the detector geometry. During 23.1 min, 83,224 frames were obtained. After background subtraction, 988 of the frames contained distinct diffraction signals of three or more Bragg spots, each corresponding to a ‘snapshot’ diffraction pattern from a different randomly oriented crystal (). Quality measures indicate that the FEL dataset conforms to diffraction data from a macromolecular crystal (Supplementary Fig. 3 and Supplementary Table 1
). In the limited time available for data collection, it was not possible to obtain a complete dataset, as indicated by the granularity in the summed powder diffraction rings () and by the statistics of the dataset (Supplementary Table 1
). As the redundancy was far too low for structure-factor extraction by Monte Carlo integration12
, an overall Rsplit
value (T.A.W. et al
., unpublished data) was not calculated. Detailed comparison to corresponding values of a similar number of diffraction patterns recorded from photosystem I nanocrystals using SFX6
showed that the data quality improves with the number of patterns, confirming that additional diffraction data will result in a complete TbCatB dataset. However, 879 (89%) of the 988 recorded diffraction patterns were indexed without unit cell constraints. The raw lattice parameters were a
= 122.9 Å, b
= 123.6 Å, c
= 53.4 Å, α = 90.3°, β = 90.2° and γ = 90.3°; therefore, the unit cell was assigned to be tetragonal, with a
= 123.3 Å and c
= 53.4 Å. Multiple measurements of individual Bragg reflections were averaged, and intensities from symmetry-equivalent reflections were merged according to the point group 4/mmm
; a pseudo-precession pattern of the  zone is shown in . Our results reveal that structural information can be obtained from in vivo
grown crystals of a glycosylated protein, providing a second independent proof of the SFX method.
Figure 2 Serial femtosecond crystallography of in vivo TbCatB crystals. (a) Diffraction pattern of a TbCatB in vivo crystal recorded from a single shot of 70 fs FEL X-rays. (b) Sum of 988 single-shot FEL diffraction patterns from TbCatB crystals in different orientations. (more ...)
In addition, PNGase F–treated TbCatB from isolated and solubilized in vivo
crystals was recrystallized in vitro
using the vapor diffusion technique. X-ray diffraction data were collected at the Swiss Light Source (Villigen, Switzerland). The structure was determined by molecular replacement using the bovine CatB structure (PDB 1QDQ) as a model and refined to 2.55-Å resolution (Supplementary Table 2
). The final structure of TbCatB (PDB 3MOR) shows the characteristic papain–cathepsin B fold, including 16 of the pro-peptide residues (Ser78 to Pro93; ), and is highly similar to a structure of nonglycosylated recombinant TbCatB that has been independently reported13
(PDB 3HHI). Here we refer to our own structural data, as we prepared our crystals from protein obtained from the in vivo
Figure 3 Structure of solubilized and recrystallized TbCatB solved by conventional X-ray crystallography. (a) Ribbon tracing of TbCatB, showing the pro-peptide in red and the occluding loop in orange. The black triangle indicates the position of the catalytic (more ...)
Despite deglycosylation treatment, our TbCatB structure still contains a glycan side chain at Asn216, clearly identified in the electron density. This suggests that, although deglycosylation was effective as judged from SDS-PAGE results (Supplementary Fig. 1c
), it removed only the glycan at Asn76. As the essential crystal contacts primarily involve hydrophobic patches within the persisting pro-peptide of TbCatB (), this pro-peptide might also influence crystal formation in vivo
). Moreover, the structure shows distinct differences from human CatB (PDB 1GMY), which are particularly important to consider for rational drug discovery investigations (Supplementary Note
The advantages of in vivo crystallization are (i) production of post-translationally modified proteins of interest, (ii) easy isolation by spinning down the crystals after cell lysis, (iii) the possibility of preliminarily analyzing crystals by EM and (iv) a narrow crystal size distribution that is ideal for SFX applications. As the use of non-insect cell signal peptides is explored further, it seems likely that more proteins will form in vivo crystals within insect cells. Recently, we obtained similar results with an inosine monophosphate dehydrogenase from trypanosomes (TbIMPDH). Under comparable experimental conditions to those reported for TbCatB, TbIMPDH in vivo crystals appeared after 5 to 6 d. Besides TbCatB and calcineurin, this is a third example of in vivo–grown crystals from a heterologously expressed protein in Sf9 insect cells. TbIMPDH crystals were isolated and subjected to a brief SFX diffraction test at LCLS. The ability of these in vivo crystals to diffract up to a resolution of more than 8 Å (data not shown) strongly supports a more general applicability of the approach described in this study.
Although progress has been made in using microfocus beamlines that allow data collection from crystals only 1 µm in size, radiation damage is an inherent problem of X-ray crystallography14
. Typically, large and well-ordered crystals of 20–500 µm are required for conventional X-ray crystallography, often a serious challenge to grow15
. Therefore, the unique combination of in vivo
crystallization and serial femtosecond crystallography offers notable new possibilities for proteins that do not form crystals suitable for conventional X-ray diffraction in vitro
, extending the available methods of structural biology particularly when, in due time, shorter wavelengths of the FEL will provide higher-resolution data.