Date of Award

December 2020

Degree Type


Degree Name

Master of Science



First Advisor

Marius Schmidt

Committee Members

Peter Schwander, Ionel Popa, Prasenjit Guptasarma


With time resolved X-ray crystallography (TRX), it is possible to follow reaction progress in real time. The time resolution is achieved by initiating reaction in crystal prior to X-ray exposure, and then collecting diffraction pattern at different time delays. Time resolved serial femtosecond crystallography (TR-SFX) at X-ray free electron laser (XFELs) allows damage free data collection from microcrystals. Mix-and-inject serial crystallography (MISC) is a type of TR-SFX established at XFELs. In MISC, reaction in enzymatic crystals is triggered by mixing with a substrate, and the resulting structural changes are probed by XFEL pulses.Enzymatic reactions are of great interest due to their biological and biomedical significance. Here we employed MISC to study the enzymatic reaction of Mycobacterium tuberculosis β-lactamase with ceftriaxone, a third-generation antibiotic. In particular we were interested in the enzyme substrate (ES) complex formation phase that triggers the catalytic reaction. We were able to follow the diffusion of substrate by structural analysis of ES complex at millisecond timescales. We also show the binding of sulbactam, an inhibitor that deactivates β-lactamase. Our results demonstrate rapid mixing experiments with MISC at XFELs is possible. It allows binding studies of ligands and drugs on other biomedically important enzymes at XFELs. This thesis is a result of my participation as a member of Prof. Schmidt’s research group in an experiment at the European XFEL (EuXFEL). Results of this experiment have been submitted to Nature in November 2020. I have been leading protein purification, crystallization and provided samples to the SPB/SFX instrument at the EuXFEL. In addition, I was participating in data collection, data analysis and data interpretation efforts of which were led by Suraj Pandey at UWM and other members of our international research team.

Available for download on Thursday, December 23, 2021

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