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Innovations in Fixed-Target Serial X-ray Crystallography
- Gilbile, Deepshika Shamraj
- Advisor(s): Kuhl, Tonya L
Abstract
Over the last decade, brilliant, coherent femtosecond X-ray free-electron lasers (XFELs) have revolutionized structural biology by enabling ultrafast, room-temperature (RT) protein structure and dynamics studies. Using a diffract-before-destroy approach, even weakly-diffracting micro or nanocrystals of hard-to-crystallize proteins can be studied using multi-crystal or “serial” data collection. But challenges exist in efficiently delivering hundreds to thousands of crystals to the X-ray beam while maintaining crystal integrity and maximizing signal-to-noise in ambient or vacuum environments. Microfabricated fixed-target supports are an exciting alternative to widely used liquid jet-based technologies as they offer distinct advantages like clog-free delivery, significantly lower sample consumption, control over sample distribution, and the ability to incorporate stimuli like ligands, caged reactants, or electric fields “on-chip” for dynamic time-resolved experiments.The works compiled in this dissertation focus on the development of two fixed-target sample delivery devices, (1) silicon micropatterned grids with ultra-thin graphene-polymer enclosing layers for structural characterization of weakly-diffracting, two-dimensional (2D) nanocrystals, or three-dimensional (3D) microcrystals where background scatter from the device can limit diffraction resolution attained, and (2) polymer microfluidic chips that enable direct on-chip crystallization and stable, long-term storage, for plug-and-play in situ diffraction measurements. Experiments on model proteins were used to benchmark the performance of these devices to demonstrate high-resolution data collection, paving the way for reliable, user-friendly protein structure studies on more scientifically interesting targets in the future.
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