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Serial Femtosecond Crystallography of Proteins in Proteins and Cancer

Description

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

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.

Contributors

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Created

Date Created
  • 2020

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Serial femtosecond crystallography data analysis of photosystem II

Description

Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a

Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually.

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.

Contributors

Agent

Created

Date Created
  • 2019