Molecular Imaging using Serial Femtosecond Crystallography

Holmes, Susannah

doi:10.26181/20001665.V1

Dissertation / PhD Thesis

PUBDB-2022-07873

Molecular Imaging using Serial Femtosecond Crystallography

Holmes, S. (Corresponding author)Extern*

2022
La Trobe

La Trobe 225 pp. (2022) [10.26181/20001665.V1] = Dissertation, La Trobe University, 2022

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Abstract: Serial femtosecond crystallography (SFX) is a technique that has allowed scientists to determine the structure of proteins to an atomic resolution using sub-micron sized crystals. Withthe development of SFX, the structural determination of crystals too small to be measured using conventional crystallography, could be solved. Using SFX, a crystal can be probed, anddiffraction obtained prior to significant damage occurring to the crystal lattice. The process of obtaining crystallography data prior to the crystal being destroyed is typically referredto as "diffraction before destruction". However, as we show in this thesis, it is possible to obtain SFX data from the same crystal more than once using consecutive X-ray pulses. Iinvestigate this phenomenon, referred to as "multi-hit" SFX using lysozyme diffraction data obtained at the European XFEL. Using the diffraction patterns obtained from only thoselysozyme crystals that had previously been probed, a second structure was solved, and after a detailed comparison it was determined that no significant radiation damage occurred. Theability to obtain a second diffraction pattern from the same crystal opens up the possibility of performing novel experiments using time-resolved SFX (tr-SFX).SFX is still a relatively new field, with continuous developments occurring. A number of different options for sample delivery exist, and part of the work in this thesis is to compareand contrast three of these sample delivery systems. This comparison used the biologically significant Myeloid differentiation primary-response 88 (MyD88) protein. The crystals wereneedle-like and were prone to aggregation which was taken into account when choosing which delivery systems were compared. Using SFX with the gas-focused dynamic virtual nozzle(GDVN), the structure of MyD88 was determined to a resolution of 2.3Å and compared to the structure of MyD88 determined via microcrystal electron diffraction (microED).

Keyword(s): Molecular Physics ; Crystallography ; 30302 Nanochemistry and Supramolecular Chemistry ; 30406 Proteins and Peptides