JINXED: Just in time crystallization for easy structure determination of biological macromolecules

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds

Abstract The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro 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 Mpro 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. Mpro 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 Mpro 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 Mpro and the crystallization conditions necessary to study this structurally