Structural biology at Sirius and the SARSCoV-2 pandemic - challenges and opportunities for global collaborations

The SARS-CoV-2 pandemic, waning now after over two years, generated a global response from the structural biology community. The first experiments at the 4th generation Synchrotron source SIRIUS, in Brazil, were focused on the structural studies of the viral proteases, including those encoded by SARS-CoV-2, their transition states and potential ligands. In this talk, we will present some of the findings concerning SARS-CoV-2 proteases and the status of MANACA beamline, as well as the latest developments in phasing (native SAD), multi-crystal, serial and room-temperature data collection. The MX beamline, MANACA, (MAcromolecular micro and Nano Serial CrystAllography), was commissioned during 2020, and the initial results helped to assess not only important features of the proteins and ligands, but also the quality and potential of the new beamline. Natural products and fragment libraries have been used by our users and collaborators [1], in academic and industrial settings. MANACA is optimised for high flux, micro-beam size and small beam divergence (0.44 mrad). Setups for serial crystallography data collection and analyses, as well as automation procedures, are being prepared [2]. The great beam characteristics provided by Sirius [3] and the high stability and precision of the optics and experimental station allows the diffraction of challenging samples such as viruses (and other crystals with large unit cells), membrane proteins and complexes, which commonly yield small crystals. The experiment control uses a userfriendly graphical interface (MXCuBE) [4], and automatic data processing (from data reduction to initial modelling) is available. The MANACA beamline is also prepared for remote access and has already performed remote experiments with foreign scientists.

A crystallographic snapshot of SARS-CoV-2 main protease maturation process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N- and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domainthree over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous Nand C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.