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- Creators: Arizona State University
photosynthesis involves the harvesting of light energy from the sun by the antenna (made
of pigments) of the PSII trans-membrane complex. The harvested excitation energy is
transferred from the antenna complex to the reaction center of the PSII, which leads to a
light-driven charge separation event, from water to plastoquinone. This phenomenal
process has been producing the oxygen that maintains the oxygenic environment of our
planet for the past 2.5 billion years.
The oxygen molecule formation involves the light-driven extraction of 4 electrons
and protons from two water molecules through a multistep reaction, in which the Oxygen
Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4.
Unraveling the water-splitting mechanism remains as a grant challenge in the field of
photosynthesis research. This requires the development of an entirely new capability, the
ability to produce molecular movies. This dissertation advances a novel technique, Serial
Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved
molecular movies may be attained. The ultimate goal is to make a “molecular movie” that
reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX)
experiments and the uniquely enabling features of X-ray Free-Electron Laser
(XFEL) for the study of biological processes.
This thesis presents the development of SFX techniques, including development of
new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL
experiment) with the goal of solving the X-ray structures in different transition states.
ii
The research comprises significant advancements to XFEL software packages (e.g.,
Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the
data acquired successfully. This research demonstrates that with manual optimizations,
the evaluation success rate was enhanced to 40-50%. These improvements have enabled
TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This
breakthrough demonstrated the first indication of conformational changes between the
ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical
predictions.
The power of the TR-SFX technique was further demonstrated with proof-of principle
experiments on Photoactive Yellow Protein (PYP) micro-crystals that high
temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved.
In summary, this dissertation research heralds the development of the TR-SFX
technique, protocols, and associated data analysis methods that will usher into practice a
new era in structural biology for the recording of ‘molecular movies’ of any biomolecular
process.
(XFEL) allows it to outrun radiation damage in coherent diffractive imaging since elastic scattering terminates before photoelectron cascades commences. This “diffract-before-destroy” feature of XFEL opened up new opportunities for biological macromolecule imaging and structure studies by breaking the limit to spatial resolution imposed by the maximum dose that is allowed before radiation damage. However, data collection in serial femto-second crystallography (SFX) using XFEL is affected by a bunch of stochastic factors, which pose great challenges to the data analysis in SFX. These stochastic factors include crystal size, shape, random orientation, X-ray photon flux, position and energy spectrum. Monte-Carlo integration proves effective and successful in extracting the structure factors by merging all diffraction patterns given that the data set is sufficiently large to average out all stochastic factors. However, this approach typically requires hundreds of thousands of patterns collected from experiments. This dissertation explores both experimental and algorithmic methods to eliminate or reduce the effect of stochastic factors in data acquisition and analysis. Coherent convergent X-ray beam diffraction (CCB) is discussed for possibilities of obtaining single-shot angular-integrated rocking curves. It is also shown the interference between Bragg disks helps ab-initio phasing. Two-color diffraction scheme is proposed for time-resolved studies and general data collection strategies are discussed based on error metrics. A new auto-indexing algorithm for sparse patterns is developed and demonstrated for both simulated and experimental data. Statistics show that indexing rate is increased by 3 times for I3C data set collected from beam time LJ69 at Linac coherent light source (LCLS). Finally, dynamical inversion from electron diffraction is explored as an alternative approach for structure determination.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
Evidence is mounting to address and reverse the effects of environmental neglect. Perhaps the greatest evidence for needing environmental stewardship originates from the ever-increasing extreme weather events ranging from the deadly wildfires scorching Greece and California to the extreme heatwaves in Japan. Scientists have concluded that the probability and severity for about two thirds of such extreme natural events that occurred between 2004 and 2018 is contributed by rising global temperatures.
Operations management literature regarding environmental issues have typically focused on the “win-win” approach with a multitude of papers investigating a link between sustainability and firm performance. This dissertation seeks to take a different approach by investigating firm responses to climate change. The first two essays explore firm emissions goals and the last essay investigates firm emissions performance.
The first essay identifies firm determinants of greenhouse gas (GHG) reduction targets. The essay leverages Behavioral Theory of the Firm (BTOF) and argues for two additional determinants, Data Stratification and Science-Based Targets, unique to GHG emissions. Utilizing system generalized method of moments on a dataset from Carbon Disclosure Project for years 2011-2017, the paper finds partial confirmation for BTOF and support for the two additional determinants of firm GHG emission goals.
The second essay is an exploratory study that seeks to understand factors for firm participation in the Science-Based Targets (SBT) initiative by combining both primary and secondary data analysis. The study is a working paper with primary data still needing to be completed. Secondary data analysis begins with a review of the literature which suggested four potential factors: ISO 14001 certification, Customer Engagement, Emission Credit Purchases, and presence of Absolute Emissions Targets. Preliminary results using panel logistic regression suggest that Emissions Credit Purchases and Absolute Emissions Targets influence SBT participation.
The third essay seeks to understand whether stakeholder pressure drives firm GHG emissions reductions. This relies on Stakeholder Theory and classification schemes proposed in Management literature to divide stakeholders, based on their relationship with the firm, into three groups: primary, secondary, and public. Random effects estimation results provide evidence for primary and public stakeholder pressure impacting firm GHG emissions.