Advances in Small-Angle X-Ray Scattering for Structural Biology at the SIBYLS Beamline
Authors:
Joshua T. Del Mundo* ([email protected]), Daniel T. Murray, Lee Joon Kim, Daniel J. Rosenberg, Brandon Russell, George Meigs, Blake Wienker, Scott Classen, David Shin, Kathryn Burnett, Susan E. Tsutakawa, Michal Hammel, Gregory L. Hura (PI)
Institutions:
Lawrence Berkeley National Laboratory
Abstract
The SIBYLS beamline conducts small-angle X-ray scattering (SAXS) to reveal the structure of biological macromolecules in solution (proteins complexes, RNA, lipid nanoparticles) and is supported by BER Integrated Diffraction Analysis Technologies (IDAT). The beamline primarily operates in high throughput (HT-SAXS) and size exclusion chromatography (SECSAXS) modes. The mail-in SAXS program offers users timely, high-quality data. Between September 2022 and September 2023, this program has resulted in 29 publications supported by IDAT. This poster summarizes some high impact studies that were made possible using the SAXS tools developed at this beamline.
First, SEC-SAXS was used in a comparative structural study to investigate the diversity in assembly of the forms of ribulose-1,5-bisphosphate carboxylase/ oxygenase (rubisco), an enzyme that catalyzes the first step in carbon fixation in plants. Characterization of the oligomeric states of deep-branching form Iα and I” rubiscos revealed structural origins of form I, shedding light onto the evolutionary path of rubisco and its transition from a homo-oligomer to a hetero- oligomer (Liu et al. 2023). Another high impact plant study investigated synthesis of pectin, an essential polysaccharide required for plant cell wall expansion. SEC-SAXS of the enzyme galactan synthase (GalS1), which catalyzes side chains in the pectin rhamnogalacturonan, was found to take on an antiparallel dimer orientation in solution, which differed from an alternative crystal structure (Prabhakar et al. 2023), giving insight into the mechanics of pectin synthesis. SAXS is also essential in understanding microbe metabolism. SEC-SAXS was used to elucidate the conformational changes within each step of electron bifurcation in EtfABCX, a membrane-bound superdimer of supermonomers (Murray et al. 2024). Electron bifurcation is a process that generates a high energy electron at the expense of the loss of energy in a second electron and is key in modelling the metabolisms of microbes for the design of novel bioenergetic systems. Finally, the team presents the utilization of SAXS as a tool in protein engineering. HT-SAXS was used to verify the architectures of various helical heterotrimer assemblies designed for use in complex protein nanostructures (Bermeo et al. 2022), as well as tunable protein crystals in solution and dried states (Li et al. 2023) for potential applications in biological sensing, catalysis, separations, and drug delivery.
After the upcoming Advanced Light Source upgrade period, the team will further improve data quality, efficiency of data collection, and implement new sample environments. Additionally, analysis pipelines are in development that will provide precise protein structural conformations by submitting sequences along with samples. The further insights that these improved SAXS studies will provide on biological macromolecules is the foundation for their applications in biomass utilization, microbe engineering, and advanced biomaterials.
References
Bermeo, S., et al. 2022. “De Novo Design of Obligate ABC-type Heterotrimeric Proteins,” Nature Structural & Molecular Biology 29(12),1266–76. DOI: 10.1038/s41594-02200879-4.
Li, Z., et al. 2023. “Accurate Computational Design of Three-
Dimensional Protein Crystals,” Nature Materials 22, 1556–63. DOI:10.1038/s41563-023-01683-1.
Liu, A. K., et al. 2023. “Deep-Branching Evolutionary Intermediates Reveal Structural Origins of Form I Rubisco,” Current Biology 33(24), 5316–25. DOI:10.1016/j.cub.2023.10.053.
Murray, D. T., et al. 2024. “Correlating Conformational Equilibria with Catalysis in the Electron Bifurcating EtfABCX of Thermotoga maritima,” Biochemistry 63(1), 128–40. DOI:10.1021/asc.biochem/3c00472.
Prabhakar, P. K., et al. 2023. “Structural and Biochemical Insight into a Modular β-1,4-Galactan Synthase in Plants,” Nature Plants 9(3), 486–500. DOI:10.1038/s41477-023-01358-4.
Funding Information
DOE BER