Date of Award


Degree Type


Degree Name

Doctor of Philosophy



First Advisor

Marius Schmidt

Committee Members

Valerica Raicu, Dilano Saldin, Arsenio Andrew Pacheco, Peter Schwander


Microcrystal, Microcrystals, Mix and Inject, Synchrotron, Time-Resolved Serial Femtosecond Crystallography, X-Ray Free Electron Laser


Time-resolved serial femtosecond crystallography (TR-SFX) employs X-ray free electron lasers (XFELs) to provide X-ray pulses of femtosecond (fs) duration with 1012 photons per pulse. These XFELs are more than a billion times more brilliant than 3rd generation synchrotron X-ray sources. For structure determination, protein crystals on the micrometer length scale (microcrystals) are injected into the X-ray beam and the resulting diffraction patterns are recorded on fast-readout pixel detectors. Although these intense pulses deposit enough energy to ultimately destroy the protein, the processes that lead to diffraction occur before the crystal is destroyed. This so-called diffraction-before-destruction principle overcomes radiation damage, which is one of the challenges that time-resolved crystallographers face at synchrotron X-ray sources. Most importantly, since each diffraction image is obtained from a fresh crystal, reversible and non-reversible reactions may be studied since both are now placed on equal footing. This is not currently possible at synchrotrons. Therefore, XFELs may provide a path forward to study reactions catalyzed by enzymes.

A TR-SFX experiment requires enormous effort and success hinges upon thorough preparation: a sufficient quantity of purified protein must be produced for the study; techniques for creating microcrystals need to be developed; these samples should then be tested with a gas dynamic virtual nozzle (GDVN) and initial studies must be performed to characterize these crystals. Since only 15% of all XFEL experiment proposals are ultimately accepted, previous results that strongly support such proposals significantly improve the chances for obtaining beamtime. I have, therefore, constructed three instruments: a micro-focus X-ray diffraction beamline, a near ultraviolet / visual wavelength fast microspectrophotometer and a GDVN fabrication and testing facility. These machines supply the crucial initial information that is needed, not only for creating engaging XFEL beamtime proposals, but also for preparing for these experiments once beamtime has been awarded.

With an initial experiment performed at the Linac Coherent Light Source (LCLS) we demonstrated for the first time that near atomic resolution time-resolved serial crystallography was possible at an X-ray FEL. This study laid the groundwork for observing the uncharacterized structures of the trans-cis isomerization of the photoactive yellow protein (PYP) photocycle on the fs timescale. Continuing on this work, we have now determined these previously unknown structures with another experiment at the LCLS. This successful fs time-resolved experiment demonstrates the full capability and vision of XFELs with respect to photoactive proteins.

In addition to studying both reversible and irreversible photo-initiated reactions, XFELs offer the unique opportunity to explore irreversible enzymatic reactions by the mix-and-inject technique. In this method, microcrystals are mixed with a substrate and the following reaction is probed by the fs X-ray pulses in a time-resolved fashion. An interesting candidate for the mix-and-inject method is cytochrome c nitrite reductase (ccNiR). This protein uses a 6 electron reduction of nitrite to produce ammonia, which is one of the key reduction processes in the nitrogen cycle. High quality large single crystals and microcrystals of ccNiR have been produced. This work is being done in collaboration with the Pacheco group in the chemistry department at the University of Wisconsin-Milwaukee. We have obtained a 1.65 Å native structure and a 2.59 Å nitrite-bound structure of ccNiR. These early studies will provide the foundation for a future time-resolved mix-and-inject XFEL proposal to study this protein.