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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., (© 2024. The Author(s).)

X-ray Free Electron Lasers (XFEL) are cutting-edge pulsed x-ray sources, whose extraordinary pulse parameters promise to unlock unique applications. Several new methods have been developed at XFELs; however, no methods are known, which allow ab initio atomic level structure determination using only a single XFEL pulse. Here, we present experimental results, demonstrating the determination of the 3D atomic structure from data obtained during a single 25 fs XFEL pulse. Parallel measurement of hundreds of Bragg reflections was done by collecting Kossel line patterns of GaAs and GaP. To the best of our knowledge with these measurements, we reached the ultimate temporal limit of the x-ray structure solution possible today. These measurements open the way for obtaining crystalline structures during non-repeatable fast processes, such as structural transformations. For example, the atomic structure of matter at extremely non-ambient conditions or transient structures formed in irreversible physical, chemical, or biological processes may be captured in a single shot measurement during the transformation. It would also facilitate time resolved pump-probe structural studies making them significantly shorter than traditional serial crystallography., (© 2024. The Author(s).)