Photosystem II (PSII) is a large protein-cofactor complex. The first step in
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.
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
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