Serial Femtosecond Crystallography of Proteins in Proteins and Cancer

This thesis focuses on serial crystallography studies with X-ray free electron lasers

(XFEL) with a special emphasis on data analysis to investigate important processes

in bioenergy conversion and medicinal applications.

First, the work on photosynthesis focuses on time-resolved femtosecond crystallography

studies of Photosystem II (PSII). The structural-dynamic studies of the water

splitting reaction centering on PSII is a current hot topic of interest in the field, the

goal of which is to capture snapshots of the structural changes during the Kok cycle.

This thesis presents results from time-resolved serial femtosecond (fs) crystallography

experiments (TR-SFX) where data sets are collected at room temperature from a

stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an

XFEL with the goal to obtain structural information from the transient state (S4)

state of the cycle where the O=O bond is formed, and oxygen is released. The most

current techniques available in SFX/TR-SFX to handle hundreds of millions of raw

diffraction patterns are discussed, including selection of the best diffraction patterns,

allowing for their indexing and further data processing. The results include two 4.0 Å

resolution structures of the ground S1 state and triple excited S4 transient state.

Second, this thesis reports on the first international XFEL user experiments in

South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this

new XFEL in a proof-of-principle experiment for the study of microcrystals of human

taspase1 (an important cancer target) by SFX has been tested. The descriptions of

experiments and discussions of specific data evaluation challenges of this project in

light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,

are included in this report

In summary, this thesis examines current techniques that are available in the

SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,

with the future potential of making movies of biological processes.