Tong You: From Photon to Protein: Using X-ray Lasers for High-resolution Structure Determination with Single Proteins

Abstract

X-ray Free-Electron Lasers (XFELs) promise Single Particle Imaging (SPI) of biomolecules through “diffraction before destruction,” but present experiments involving single proteins are limited by weak scattering signals and significant background scattering. This background scattering comes primarily from the sample delivery, and poses a major challenge for small proteins. Recent progress in aerosol sample delivery has shown that the background scattering can be reduced by about 80% by replacing some of the carrier gas with helium. The first paper in this thesis was a large-scale simulation study of a 15 nm diameter GroEL protein, where the impact of reduced gas background on the resolution was investigated. In Paper II the feasibility of SPI is investigated using liquid sheet sample delivery with the same protein. I find that it is indeed possible to do SPI in solution but with demanding requirements: specifically, a large number of diffraction patterns and a high fluence, which currently only a nanofocus can deliver. The second half of this thesis focuses on my contribution to analysis performed on experimental data. Paper III is about work where I developed the initial scripts for data analysis of a Rayleigh-scattering microscope used to characterise the aerosol sample delivery system that is also used during SPI experiments. The final paper is about my contribution to an experiment where the liquid sheet sample delivery was used during an experiment. I end this thesis with an outlook where I provide my perspective on the computational and experimental improvements that can make the biggest impact on the future of SPI.