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.
Highlights
During the past two decades, macromolecular X-ray crystallography (MX) has been revolutionized by the establishment of high-brilliance radiation sources and novel developments in serial diffraction data-collection strategies (Standfuss & Spence, 2017; Yamamoto et al, 2017; Yabashi & Tanaka, 2017; Spence, 2020)
Based on the ultrashort X-ray pulses that diffract at high resolution even from crystals with dimensions in the submicrometre size range, this serial femtosecond X-ray crystallography (SFX) approach provides new opportunities for structural biology (Johansson et al, 2017), including time-resolved X-ray diffraction to visualize the molecular dynamics of macromolecules by pump–probe experiments (Kupitz et al, 2014; Tenboer et al, 2014; Shimada et al, 2017; Nango et al, 2016)
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 fixed-target SFX (FT-SFX) of intracellular crystals at 1.8 Aresolution from a single chip (PDB entry 7asx)
Summary
During the past two decades, macromolecular X-ray crystallography (MX) has been revolutionized by the establishment of high-brilliance radiation sources and novel developments in serial diffraction data-collection strategies (Standfuss & Spence, 2017; Yamamoto et al, 2017; Yabashi & Tanaka, 2017; Spence, 2020). The extremely short femtosecond pulses generated by X-ray free-electron lasers (XFELs) outrun radiation-damage effects, which limit the usable dose at synchrotron sources, enabling almost damage-free diffraction data collection at room temperature following the ‘diffractionbefore-destruction’ approach (Neutze et al, 2000; Chapman et al, 2011). This major challenge in XFEL diffraction experiments has been overcome in recent years by advancements in crystal-delivery strategies (Martiel et al, 2019). Sequential crystal-delivery strategies have successfully been adapted for serial synchrotron data collection to reduce dose accumulation in a single crystal, advancing the application of serial synchrotron crystallography (SSX) on microcrystals even at room temperature (Gati et al, 2014; Stellato et al, 2014; Roedig et al, 2016; Owen et al, 2017; Weinert et al, 2017; Schulz et al, 2018; Mehrabi et al, 2019)
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