Vallejos, Adams2024-11-222024-11-222024-11-22978-91-8069-964-8 (PDF)978-91-8069-963-1 (tryckt)https://hdl.handle.net/2077/83570Many transmembrane proteins play an essential role by facilitating energy transduction in various biological systems. Whether powered by light, as in bacteriorhodopsin or photosynthetic reaction centres, or by oxygen reduction, as in cytochrome c oxidase, these proteins enable directional proton transport across the membrane. For bacteriorhodopsin and cytochrome c oxidase, this is achieved through structural changes that alternate the accessibility of their active sites to either side of the membrane, and for photosynthetic reaction centres through redox reactions coupled to electron movements across the membrane. These mechanisms allow these proteins to establish electrochemical gradients crucial for cellular functions including ATP synthesis, photosynthesis, and cellular respiration. In this research, we use time-resolved serial crystallography to resolve the three-dimensional structures of these three molecular systems and investigate the time-dependent conformational changes they undergo after light excitation. We developed and employed a variety of computational methods to characterize protein dynamics using statistics. We investigate the role of occupancy in popular structural refinement strategies, namely partial-occupancy refinement or refinement against extrapolated data to assess the structural shifts associated with light activation events. In addition, we integrate these structural refinement strategies with resampling methods to estimate coordinate uncertainties, ensuring robust and reliable interpretation of these transient states. To further analyze structural dynamics, we use difference Fourier maps, which minimize phase bias and enhance our ability to resolve subtle but functionally significant changes, and employ singular value decomposition to analyze them in two ways: First, as a tool for enhancing the signal-to-noise of these maps, and second, in combination with resampled maps to improve our confidence while assigning time-dependent difference density features. We also examine residual densities for transient water molecules by comparing difference Fourier and Polder omit maps, allowing us to characterize transient hydration states involved in the energy transduction mechanisms. Together, these methods provide a comprehensive framework for elucidating the precise structural dynamics in these proteins, advancing our understanding of how energy transduction is coordinated at the molecular level. These approaches offer valuable insights into the mechanisms underlying key biological processes, from photosynthetic energy capture to cellular respiration.engStructural biologyMembrane proteinsBacteriorhodopsinCytochrome c oxidaseReaction centreSerial X-ray crystallographyTime-resolved X-ray crystallographyProtein dynamicsCoordinate errorsResamplingText