Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

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.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

“…In fixed-target methods, the crystal suspension is loaded onto the surface of a solid support that is rastered through the interaction region of the X-ray beam. These devices use thin layers of materials such as silicon, 38,39 cycloolefin-copolymer (COC), 40,41 polydimethylsiloxane (PDMS), 42 COC/PDMS combinations, 43 polyethylene terephthalate, 44 graphene layers in combination with polymers, 45 and polyimide. 46 Typically, fixed-target devices enable high hit rates (10–40%) compared to the lower hit rates (1–10%) frequently achieved with continuous liquid delivery systems.…”

Convolutional neural network approach for the automated identification ofin cellulocrystals

“…The vast majority of the crystals tend to place at the bottom of the cells, as observed with confocal optical sectioning (Figure 2). HEX-1 crystals have been observed in the baculovirus-infected cells at day 4 p.i., exhibiting the reported hexagonal shape and the expected crystallization efficiency (>85 %), without protruding the cell body (Lahey-Rudolph et al, 2021). Next to the smaller size, HEX-1 crystals show a significant degree of clustering and grow in diverse orientations (Figure 3), adding a level of complexity to crystal analysis compared to GMPR, for both manual annotation and automatic segmentation.…”

“…To date, around 80 targets have been found to crystallize in different cells (Tsukimoto et al, 2022; Li et al, 2020; Nass et al, 2020; Mudogo et al, 2020; Schönherr et al, 2018; Duszenko et al, 2015; Koopmann et al, 2012). Some of them were successfully used as targets for protein structure elucidation (Redecke et al, 2013; Baskaran et al 2015; Tsutsui et al 2015; Nass et al, 2020; Lahey-Rudolph et al, 2021). Intracellular protein crystallization can occur naturally, providing distinct advantages for the cell (Schönherr et al, 2018), or as a consequence of heterologous gene expression in host cells.…”

“…We recently reported that spontaneous self-assembly of HEX-1 into intracellular crystals is not restricted to the native environment of fungal cells (Lahey-Rudolph et al, 2020). On infection with a recombinant baculovirus encoding the HEX-1 gene, living insect cells also form regular, micrometre-sized hexagonal crystals that enabled structure elucidation using the fixed-target serial femtosecond crystallography approach (FT-SFX) at an XFEL (Lahey-Rudolph et al, 2021).…”

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

“…Fortunately, smaller crystal size requirements and the ability to deliver crystals at room temperature have opened the door to new sample delivery methods for XFEL-based crystallography ( 52 ). Delivery of crystals on solid supports, also known as “fixed targets,” is one of the approaches whereby sample crystals are loaded on a matrix or a support device made from highly X-ray transparent materials such as silicon chips ( 53 , 54 ), thin membranes ( 55 , 56 ), elastomers ( 57 ), and thermoplastic materials ( 58 , 59 ), enabling in some cases more than 100% hit rates (multiple crystals per shot) as opposed to the typically low hit rates (1%–10%) currently achievable with continuous liquid delivery systems ( 6 , 60 , 61 , 62 ). Recently, Shelby et al.…”