Lars Redecke

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. In 2024, the InCellCryst pipeline was introduced for structural studies of intracellular crystals by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution studies on proteins that crystallize only in a few cells. Here we introduce IncelluloED , a method that combines intracellular crystallization with in situ 3D electron diffraction in cells and achieves high-resolution structures from just one crystal inside one cell. Experiments on a microcrystal of the HEX-1 protein from Magnaporthe grisea , grown inside an insect cell, give a structure at 1.9 Å resolution from a volume of ~1.6 µm ³ as compared to 1.8 Å resolution achieved by serial X-ray crystallography from a combined volume exceeding eleven million µm ³ . IncelluloED uses widely available cryo-EM tools and brings high-resolution structural biology into home laboratories while also advancing a vision for a “single-cell structural laboratory”.

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. An InCellCryst pipeline was recently established for structure determination by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution structural studies on proteins with low numbers of crystals. To overcome this limitation, we combined the InCellCryst approach with the advantages of 3D electron diffraction and established the new IncellED method. Using microcrystals of the HEX-1 protein from Magnaporthe grisea , grown inside High Five insect cells, we demonstrate that electron diffraction data collected from a single crystal yields a high-resolution structure at 1.9 Å resolution, comparable to 1.8 Å achieved by serial X-ray crystallography. The IncellED methodology opens structure determination for proteins that crystallize with low efficiency and use widely available electron cryo-microscope infrastructure, paving the way for intracellular macromolecular electron crystallography at high resolution.

Crystallization of recombinant proteins in living cells is an emerging approach complementing conventional crystallization techniques. Homogeneous microcrystals well suited for serial diffraction experiments at X‐ray free‐electron lasers and synchrotron sources can be produced in a quasi‐native environment, without the need for target protein purification. Several protein structures have already been solved; however, exploiting the full potential of this approach requires a systematic and versatile screening strategy for intracellular crystal growth. Recently, we published InCellCryst, a streamlined pipeline for producing microcrystals within living insect cells. Here, we present the detailed protocol, including optimized target gene expression using a baculovirus vector system, crystal formation, detection, and serial X‐ray diffraction directly in the cells. The specific environment within the different cellular compartments acts as a screening parameter to maximize the probability of crystal growth. If successful, diffraction data can be collected 24 days after the start of target gene cloning.

The main protease (M^pro) of SARS-CoV-2 is critical for viral function and a key drug target. M^pro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized M^pro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. M^pro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized M^pro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of M^pro and the crystallization conditions necessary to study this structurally.

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by in vitro crystallization extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely native proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, improved strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

In cellulo crystallization is a rare event in nature. Recent advances that have made use of heterologous overexpression can promote the intracellular formation of protein crystals, but new tools are required to detect and characterize these targets in the complex cell environment. The present work makes use of Mask R-CNN, a convolutional neural network (CNN)-based instance segmentation method, for the identification of either single or multi-shaped crystals growing in living insect cells, using conventional bright field images. The algorithm can be rapidly adapted to recognize different targets, with the aim of extracting relevant information to support a semi-automated screening pipeline, in order to aid the development of the intracellular protein crystallization approach.

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by conventional methods extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely unmodified proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, new strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

Nipah virus (NiV) is a zoonotic paramyxovirus with a fatality rate of up to 92% in humans. While several pathogenic mechanisms used by NiV to counteract host immune defense responses have been described, all of the processes that take place in cells during infection are not fully characterized. Here, we describe the formation of ordered intracellular structures during NiV infection. We observed that these structures are formed specifically during NiV infection, but not with other viruses from the same Mononegavirales order (namely Ebola virus) or from other orders such as Bunyavirales (Junín virus). We also determined the kinetics of the appearance of these structures and their cellular localization at the cellular periphery. Finally, we confirmed the presence of these NiV-specific ordered structures using structured illumination microscopy (SIM), as well as their localization by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and correlative light and electron microscopy (CLEM). Herein, we describe a cytopathogenic mechanism that provides a new insight into NiV biology. These newly described ordered structures could provide a target for novel antiviral approaches.

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.