Design of a Micro-Focus X-Ray Source Validated by Model Protein Crystals

The main objective of this thesis was to build a Laboratory Microfocus X-ray Source for Protein Crystallography (LMXS-PC), an X-ray source that would provide prospective users programmatic control and remote capability. A study of the crystal was conducted using Photoactive Yellow protein (PYP) and cytochrome c nitrite reductase (ccNiR). The instrument was then tested by mounting the crystal in the instrument and collecting data. The other objective of my research work was to determine the crystal structure of PYP using X-ray crystallography techniques. I attempted many trials in PYP crystal in an effort to achieve a high-quality diffraction pattern; however, crystals diffract to less than 2 Å resolution limit. My interest in the crystal of Photoactive Yellow Protein to understand how electron density is obtained from the information contained within the diffraction patterns

REdiii: a pipeline for automated structure solution

High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort. REdiii is a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies in case the preconfigured solution is inadequate. This integrated pipeline is targeted at providing a comprehensive and efficient approach to screening for ligand-bound co-crystal structures while minimizing repetitiveness and allowing a high-throughput scientific discovery process

Tightly integrated single‐ and multi‐crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109583/1/S1600576714022730.pd

Data Publication with the Structural Biology Data Grid Supports Live Analysis

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis

Data Publication with the Structural Biology Data Grid Supports Live Analysis

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis

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

47, 1992-1999 Sudhir Babu Pothineni et al. · JBluIce beamline control system research papers

This open-access article is distributed under the terms of the Creative Commons Attribution Licence http://creativecommons.org/licenses/by/2.0/uk/legalcode, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. Many research topics in condensed matter research, materials science and the life sciences make use of crystallographic methods to study crystalline and non-crystalline matter with neutrons, X-rays and electrons. Articles published in the Journal of Applied Crystallography focus on these methods and their use in identifying structural and diffusioncontrolled phase transformations, structure-property relationships, structural changes of defects, interfaces and surfaces, etc. Developments of instrumentation and crystallographic apparatus, theory and interpretation, numerical analysis and other related subjects are also covered. The journal is the primary place where crystallographic computer program information is published. Crystallography Journals Online is available from journals.iucr.org J. Appl. Cryst. (2014 The calculation of single-and multi-crystal data collection strategies and a data processing pipeline have been tightly integrated into the macromolecular crystallographic data acquisition and beamline control software JBluIce. Both tasks employ wrapper scripts around existing crystallographic software. JBluIce executes scripts through a distributed resource management system to make efficient use of all available computing resources through parallel processing. The JBluIce single-crystal data collection strategy feature uses a choice of strategy programs to help users rank sample crystals and collect data. The strategy results can be conveniently exported to a data collection run. The JBluIce multi-crystal strategy feature calculates a collection strategy to optimize coverage of reciprocal space in cases where incomplete data are available from previous samples. The JBluIce data processing runs simultaneously with data collection using a choice of data reduction wrappers for integration and scaling of newly collected data, with an option for merging with pre-existing data. Data are processed separately if collected from multiple sites on a crystal or from multiple crystals, then scaled and merged. Results from all strategy and processing calculations are displayed in relevant tabs of JBluIce

Structural and kinetic studies of bifunctional and monofunctional proline catabolic enzymes

Title from PDF of title page (University of Missouri--Columbia, viewed on December 4, 2015).PutAs, Proline dehydrogenase and P5C dehydrogenase are involved in the oxidation of proline to glutamate. Mutations in proline dehydrogenase and P5C dehydrogenase cause a disease condition known as hyperprolinemia. In this work I have solved the crystal structure of bifunctional PutA and P5C dehydrogenase. The structure of P5C dehydrogenase sheds light on the structural basis of hyperprolinemia. I have also used kinetic and thermodynamic methods to understand the molecular mechanism of hyperprolinemia