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. 2021 Jun 18;8(Pt 4):665-677.
doi: 10.1107/S2052252521005297. eCollection 2021 Jul 1.

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

J Mia Lahey-Rudolph  1   2 , Robert Schönherr  1   3 , Miriam Barthelmess  2 , Pontus Fischer  2 , Carolin Seuring  2   4 , Armin Wagner  5 , Alke Meents  2   3 , Lars Redecke  1   3
Affiliations

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

J Mia Lahey-Rudolph et al. IUCrJ. .

Abstract

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Keywords: Roadrunner; fixed-target SFX; in cellulo crystallography; intracellular protein crystals; serial femtosecond crystallography; silicon chip.

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Figures

Figure 1
Figure 1
Crystallization of HEX-1 from N. crassa in living insect cells. Light-microscopic images of Sf9 cells four days after infection with a recombinant baculovirus encoding the target protein (MOI = 1). Most cells produce a single crystal per cell (a). Two different crystal morphologies can appear. The first is a spindle-like morphology with flat ends and a slightly increased diameter in the middle (b) showing a hexagonal cross section (c). These crystals sometimes grow in a star-like manner, conjoined in the middle (d). The second morphology is bipyramidal (e) with pointed tips and a square base (f). These crystals can sometimes form somewhat irregular forms with rounded tips (g). Both morphologies can grow cytoplasmatically as well as within the cell nucleus [(b) versus (h) and (e) versus (i)]. After cell lysis the crystals show high stability in the cell-culture medium, while mostly sticking to cell remnants (j). Bipyramidal crystals constitute about 13% of the total crystal population. The size distribution shows that the bipyramidal crystals form shorter but broader crystals overall compared with the spindle-like crystals (k). Size bars: 50 µm (a), 10 µm (b)–(j).
Figure 2
Figure 2
(a) An empty micro-patterned silicon chip designed for the Roadrunner II goniometer. The chip contains 12 µm sized pores that are spaced 90 and 100 µm apart, allowing well defined positioning of crystal-containing cells. (b) Sf9 insect cells containing in cellulo grown HEX-1 crystals were sucked into the chip pores. Visible intracellular crystals positioned in the pores are marked with yellow arrows; blue arrows point into empty pores. The images were recorded using reflected light on an Olympus SZX16 microscope.
Figure 3
Figure 3
(a) An individual diffraction pattern of an intracellular HEX-1 crystal in a Sf9 cell, recorded at room temperature on a Roadrunner II chip with a CSPAD detector. The maximum resolution was limited by the setup to 2.0 Å at the detector edges. Higher resolution peaks of up to 1.70 Å could be detected in the detector corners (blue inset). The shape of the peaks indicates low mosaicity (red inset). (b) Virtual powder diffraction pattern of HEX-1 crystals recorded in cellulo shows a homogeneous distribution of crystal orientations. The sum of detected Bragg peaks detected by indexamajig from the CrystFEL suite in all images resulting from the data collection from chips 1 and 2 is depicted.
Figure 4
Figure 4
Details of the HEX-1 structure. (a) Overall structure of HEX-1 in cartoon representation. (b) Representative region of the electron-density map of HEX-1 obtained by FT-SFX of intracellular crystals at 1.8 Å resolution from a single chip (PDB entry 7asx). The detailed region is marked by a black box in (a). (c) Structural homology of HEX-1 crystallized in insect cells and HEX-1 purified from E. coli and crystallized by sitting-drop vapour diffusion. A backbone representation of the HEX-1 structure (green) obtained from in cellulo diffraction is superimposed with that of the HEX-1 reference structure (PDB code 1khi; blue). The average r.m.s.d. is 0.47 Å for equivalent Cα atoms. The only region showing major structural differences with r.m.s.d.s above 0.6 Å is highlighted by the red box. (d) OMIT map of residues 62–65 of the FT-SFX HEX-1 structure with the same residues in stick representation. The F o − F c map (green) of the region of largest r.m.s.d.s with the reference structure, highlighted in (c), is contoured at 3σ.
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Grants and funding

This work was funded by Bundesministerium für Bildung und Forschung grant 05K18FLA to Lars Redecke; Joachim Herz Stiftung; Deutsche Forschungsgemeinschaft.