Lavens, A. | bioRxiv Science

bioRxiv2022

New system for serial crystallography data collection at SBC APS

Serial synchrotron crystallography enables studies of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed by using a high-performance computing pipeline. Here, the system was applied to two {beta}-lactamases: a class D serine {beta}-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-{beta} -lactamase from Stenotrophomonas maltophilia K279a.

molecular biology↗

bioRxiv2020

Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5' terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2'-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2'-O Cap-1, where 2'OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

molecular biology↗