Abstract
Hydrogen atoms represent a large fraction of the total atomic content of macromolecules. They often play critical roles in enzyme catalysis, ligand recognition processes, and protein-protein interactions. However, their direct visualisation by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localisation of only the most ordered hydrogen atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many hydrogen atoms of biochemical significance remain undetectable by this method. Differently, neutron diffraction methods enable the visualisation of most hydrogen atoms, typically in the form of deuterium (2H) atoms at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought.The main objective of this PhD project was to develop novel methods and tools to enhance the refinement of macromolecular models utilising neutron crystallographic data. Novel refinement protocols have been implemented in the macromolecular refinement software REFMAC5, one of the flagship packages of the Collaborative Computational Project Number 4 (CCP4) suite of programs.
This thesis is divided in five chapters. Chapter 1 provides an initial overview of neutron macromolecular crystallography, emphasizing its significance in the field of structural biology.
Chapter 2 is divided in two parts. The first part provides an overview of macromolecular refinement, highlighting capabilities within REFMAC5 and exploring current neutron refinement strategies in other software packages. The second part of this chapter describes the novel neutron refinement implementation, focusing on the treatment of hydrogen atoms during the refinement process within CCP4.
Chapter 3 presents the re-refinement of several entries available in the Protein Data Bank (PDB) by using either (i) neutron data-only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. A comparative analysis was conducted between refinement statistics obtained from previously employed software packages and those obtained from REFMAC5. Re-refinement with REFMAC5 afforded models characterised by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium-low resolution. In addition, a detailed example of neutron re-refinement of the structure of chloride-free urate oxidase (UOX) in complex with its 8- azaxanthine (8AZA) inhibitor is presented, confirming the protonation states of active site residues involved in ligand interactions, thereby contributing to elucidating the hydration step of UOX catalysis.
Chapter 4 introduces the refinement of two novel neutron crystallographic structures using REFMAC5. The initial case delves into the neutron refinement process applied to perdeuterated UOX complexed with the 5- peroxo derivative of 9-methyl uric acid (9-MUA). This chapter also offers an overview of the UOX enzyme, highlighting its clinical significance, along with a discussion on the structural and mechanistic challenges associated with it. The neutron refinement of the UOX:5-PMUA complex performed with REFMAC5 confirmed the presence of the trapped peroxide intermediate. Furthermore, the examination of protonation states of the catalytic residues offers valuable insights into the enzyme mechanism. The structure presented in this chapter is discussed with reference to the current knowledge of the UOX mechanism of action, providing an exciting platform for future work into the structure of reactive intermediates in UOX catalysis. The second example shows the results from the neutron refinement of Prochlorococcus iron binding protein FutA. These investigations have revealed that FutA is capable to accommodate the binding of iron in the ferric (Fe3+) state, which was previously challenging to detect due to X-ray-induced photoreduction. This represents the first neutron structure refined using REFMAC5 and deposited in PDB under accession number 8RK1.
| Date of Award | 1 Apr 2024 |
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| Original language | English |
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| Supervisor | Roberto Steiner (Supervisor) & Garib N. Murshudov (Supervisor) |