Time-Resolved, Near Atomic Resolution Structural Studies at the 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

Verification of a quantitative model to characterize granular flow - A measure of mixing of grain layers

Traceability has been an area of much research in recent years due to the need for food quality and safety and the subsequent regulations and legislation that have been put in place around the world. There are economic and market advantages that can be gained from industry members that put traceability tools to use effectively. Bulk products challenge traceability efforts more than other foods due to the complications presented by: 1) bulk storage of many source lots in one container; 2) granular flow characteristics influenced by the grain and the container; and 3) commingling practices that make exact composition of lots difficult to produce. Much is known about granular flow due to the importance of granular materials in industries around the world. Granular flow produces different flow regimes, or behaviors, under different conditions, and many of those conditions have been tested. There is however, a lack of research into what those flow regimes mean for granular mixing as grain moves through a storage container filled with multiple source lots. This experiment is the beginning of developing that understanding. It consisted of a small model layered with easily differentiable source lots comprised of granular materials that are the same size, shape and mass. Glass beads in easily identifiable colors were used to address the need for uniformity and to provide differentiation. The material is then drawn from an opening in the floor of the apparatus, as it would be in a grain facility, the layers sorted and weighed, and the mixing quantified. Much more work will be needed in this area of research, but the results of this experiment are promising for development of probability models to describe the composition of grain shipments

A quantitative model to characterize granular flow behavior – A measure of grain layer mixing in storage facilities

Food quality and safety concerns have led to many nations implementing regulation and legislation around acceptable food and feed practices. Traceability systems are one component of food and feed practices that have been a topic of much research in recent years. Traceability tools are useful for ensuring regulations are met and offering metrics for improving company processes. Companies that implement traceability systems reap economic benefits, gain market advantage, and decrease losses from costly food recalls through the ability to more efficiently remove contaminated food from the value chain. Bulk commodities, such as grain, greatly increase the difficulty inherent in designing and implementing a traceability system. Comingling grain from various sources is a common practice to transform grain of various quality attributes to achieve an overall higher quality grain for sale to processors. Comingling complicates traceability as granular flow combines all grain sources together with no clear separation point. Previous granular flow research shows that there are two main flow regimes present in a granular material flowing in this manner: 1) Mass flow, where all of the material is in motion and the grain is removed in a mostly first-in-first-out (FIFO) behavior, and 2) Core flow, where the grain forms a natural hopper with some of the grain forced into stagnation and providing a mostly last-in-first-out (LIFO) behavior. The amount of mixing that occurs due to the layering of grain and the flow regimes present as grain is removed has not been previously quantified. Assumptions are made based on the FIFO and LIFO flow regimes but result in a lack of certainty about shipping container composition after the grain is removed from the bin. This lack of certainty leads to costly and inefficient recalls. This experiment is a first step in the development of understanding how much mixing is occurring in grain storage bins. It consisted of the design and development of a small model similar in structure to a flat-floored cylindrical grain bin. The experimental model presented flow behavior that aligned with expected regimes for flat-floored structures and provided consistent data. These outcomes signify that the model and the method are not causing significant changes in flow behavior and indicate that further testing and scaling should be possible. Outcomes from this quantification of granular mixing will provide a useful tool in the area of traceability. Given enough data on the mixing between layers of various types of grain probability models can be developed to provide a more precise prediction of what the composition of each shipment consists of on a percent basis

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium

Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

Biomolecular simulation is increasingly central to understanding and designing biological molecules and their interactions. Detailed, physics‐based simulation methods are demonstrating rapidly growing impact in areas as diverse as biocatalysis, drug delivery, biomaterials, biotechnology, and drug design. Simulations offer the potential of uniquely detailed, atomic‐level insight into mechanisms, dynamics, and processes, as well as increasingly accurate predictions of molecular properties. Simulations can now be used as computational assays of biological activity, for example, in predictions of drug resistance. Methodological and algorithmic developments, combined with advances in computational hardware, are transforming the scope and range of calculations. Different types of methods are required for different types of problem. Accurate methods and extensive simulations promise quantitative comparison with experiments across biochemistry. Atomistic simulations can now access experimentally relevant timescales for large systems, leading to a fertile interplay of experiment and theory and offering unprecedented opportunities for validating and developing models. Coarse‐grained methods allow studies on larger length‐ and timescales, and theoretical developments are bringing electronic structure calculations into new regimes. Multiscale methods are another key focus for development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology. This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Free Energy Method