Macromolecular Structure Determination at X-Ray Free Electron Lasers from Single-Particle Imaging to Time-Resolved X-Ray Crystallography
Date of Award
Doctor of Philosophy
Peter Schwander, Abbas Ourmazd, Valerica Raicu, Ionel Popa
X-ray free-electron lasers (XFELs) open the possibility of obtaining diffraction information from a single biological macromolecule. This is because XFELs can generate extremely intense X-ray pulses which are so short that diffraction data can be collected before the sample is destroyed. By collecting a sufficient number of single-particle diffraction patterns from many tilts of a molecule relative to the X-ray beam, the three-dimensional electron density can be reconstructed ab-initio. The resolution and therefore the information content of the data will ultimately depend largely on the number of patterns collected at the experiment. We estimate the number of diffraction patterns required to reconstruct the electron density at a targeted spatial resolution. This estimate is verified by simulations for realistic X-ray fluences, repetition rates, and experimental conditions available at modern XFELs. Employing the bacterial phytochrome as a model system we demonstrate that sub-nanometer resolution is within reach.
Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. Attaching biological molecules to highly scattering AuNPs may provide a powerful way to identify and resolve single molecules. Here, we have shown the weakly scattering part, the thiol coating can be resolved in the presence of a strong scatterer gold from a thiol-decorated gold nanoparticle using the single-particle imaging (SPI) method. One of the scientific cases for building XFELs is the structure determination of single macromolecules and macromolecular complexes at atomic resolution. We propose a promising route of obtaining a sub-nanometer resolution to visualize the atomic details of biological macromolecules using SPI with XFELs.
Biological processes are highly dynamic. Static structures may provide limited insight into protein function, but they offer no information on protein dynamics. For understanding how proteins function, one must investigate structural changes as they happen. The advent of the first XFELs has enabled time-resolved serial femtosecond crystallography (TR-SFX) experiments. The reaction is initiated by light excitation or by the rapid mixing of the microcrystals of functionally active biomolecules. The structural changes are probed after a certain time-delay by the fs X-ray pulses. The European XFEL (EuXFEL), with its unique pulse structure, opens a new era for TR-SFX in the study of the dynamics of biological systems. At EuXFEL, X-rays arrive in pulse trains at 10 Hz. When the design specifications are reached, it can produce up to 2,700 pulses with up to 4.5 MHz pulse repetition in a train. At 4.5 MHz, each pulse train is 600 µs long with nearly 99.4 ms gaps between the trains. The ultrashort pulse length of the EuXFEL gives the unique possibility to study the dynamics of very short-lived complexes and transient states of dynamic molecules.
We have conducted a first time-resolved experiment using PYP as a model system with a complex MHz X-ray pulse structure at the EuXFEL. This successful time-resolved experiment opens the door for future time-resolved experiments at the EuXFEL. With the knowledge and experience gained from TR-SFX experiments, the possibility of a similar experiment with time-resolved SPI in the future is also discussed.
Poudyal, Ishwor, "Macromolecular Structure Determination at X-Ray Free Electron Lasers from Single-Particle Imaging to Time-Resolved X-Ray Crystallography" (2020). Theses and Dissertations. 2580.