Works on reconstitution of multi-protein complexes (MPCs) for biochemical and X-ray crystallographic studies. In particular we are interested in MPCs involved in: 1) nuclear events such as transcription initiation and DNA repair, and 2) Interactions of membrane receptors with their cytoplasmic partners. Since MPCs cannot be isolated as a whole from cells, we have developed biochemical tools that have allowed us to express and purify individual components (monomeric or multimeric) to reconstitute such MPCs. Our ultimate goals is to crystallize and perform structural studies using X-ray crystallography of these samples which posses great biological interest. We are also participating in development of methods using EM to identify best diffracting nano-crystals for femtosecond diffraction experiments.
Research in Dr. Fraser’s lab focuses on how protein conformational ensembles respond to perturbations such as temperature, ligand binding, and mutation. The lab develops new data collection capabilities (with a focus on ambient temperature data collection and diffuse scattering) and creates new multiconfomer representations of protein structure and dynamics. Within the BioXFEL center, Dr. Fraser’s group is interested in novel electron density analyses of time resolved data and studying the differences in radiation damage between synchrotron and XFEL data sets.
As part of the BioXFEL Center, Dr. Fromme's group focuses both on method development of fs crystallography and the resolution of important biological questions using the novel methods developed. The project on method developments includes novel strategies and optimization of methods for growth, detection and characterization of protein nanocrystals. Trained as a biophysical chemist, she uses a rational design strategy that includes phase diagram determination for growth and size-stabilization of nanocrystals in high throughput. A large range of biophysical methods are currently used and under further development to characterize nanocrystals including, powder diffraction methods, dynamic light scattering and Second Order Non-linear Imaging of Chiral Crystals ( SONICC). Another focus for the BioXFEL Center of the Fromme Lab will be the development of improved methods for sample delivery that work in high throughput with minimal sample use in collaboration with the team at ASU. Her group also works with the team at ASU, at Stanford and Dr. Chapman at DESY on development of new methods for data evaluation for fs nanocrystallography data. This includes the development of new algorithms for indexing and structure factor refinement as well as phase determination.
Dr. Hogue's Lab will develop approaches and optimization for single-particle imaging of virus particles for structure determinations using BioXFELS. They will also work on production and imaging of nanocrystals of viral proteins for structural reconstructions. Their work is targeting collaboration with John Spence, Uwe Weierstall, Bruce Doak, Petra Fromme, Dilano Saldin and Abbas Ourmazd. https://sols.asu.edu/people/brenda-hogue
The Kornberg lab's general interest in the BioXFEL Center is to explore the use of the nanocrystals and single particle analysis to study transcription and transcription regulator complexes. The Kornberg lab has two immediate projects they will embark on via the BioXFEL Center. The first project is a serial femtosecond crystallography project on the 1 MDa mediator complex for which they have nanocrystals, but have not demonstrated diffraction beyond ~20angstroms. They have PCS tested the diffraction of the Mediator crystals during LCLS run8. The second project is a time resolved study of transcription elongation. They have solved via traditional MX methods four stable states of RNA Polymerase II during transcription, and would like to fill in the transitions between these stable states using time resolved XFEL. A millisecond long Markov State Model simulation of the elongation process indicates there are two new metastable intermediate states that they have not been able to access using tradition MX methods. The Kornberg Lab plans to explore the use of caged compounds, as well as the proposed mixing injectors for this study. http://kornberg.stanford.edu/
Dr. Ourmazd's effort with the BioXFEL Center is directed at determining the structure, conformations, and dynamics of biological entities such as viruses and proteins, and molecular machines, such as the ribosome.
Ourmazd develops advanced algorithms to reconstruct the three-dimensional structure of non-stationary objects from random, ultra-low-signal sightings of unknown orientation. Applications range from single biomolecules and viruses to non-stationary macroscopic objects such as breathing lungs and beating hearts. More generally, his techniques offer novel search algorithms for furtive signals in a sea of noise.
Dr. Phillips' research is focused primarily upon computational biology and examining the structure and dynamics of proteins. Other ongoing projects in his lab include, acquiring an atomic description of the basis for binding oxygen and other ligands to heme proteins; developing new techniques for observing the dynamics of proteins and nucleic acids using diffuse X-ray scattering analysis and molecular dynamics simulations; structural genomic research, and a joint research venture with the Great Lakes Bioenergy Research Center focused upon biofuel, with accentuation on cellulosic ethanol development. http://phillipslab.org/
Lois Pollack's research program has two distinct themes. The first theme is instrumentation: the development of experimental tools that enable novel, time-resolved studies of proteins, DNA or RNA. By coupling microfluidics with light (either X-rays or lasers). Her group has developed and applied tools that report dynamic shape changes as these large molecules assume ('fold' to) their biologically active states. The second theme is a tight research focus on electrostatic interactions in RNA and DNA. The large negative charge carried by these nucleic acids significantly impacts their structure and function. This topic is timely, as recognition of RNA's central role in the cell continues to increase at an astonishing rate.
As part of the BioXFEL Center, the Pollack Lab, which specializes in developing new instrumentation to advance small angle X-ray scattering studies of proteins, RNA and DNA will focus its efforts on time-resolved SAXS as a probe of conformational dynamics and folding. In the past, they applied microfluidic mixers to trigger and monitor protein and RNA folding using SAXS. The group has also developed a microfluidic cell to trigger and measure light-dependent protein conformational changes. X-ray free electron laser sources present new opportunities for determining solution structures of bio-molecules based on analyzing angular correlations within individual SAXS profiles. Their successful demonstration of these new methods will enable time-resolved studies of structural changes, initiated either by laser flash, or by rapid solvent exchange. http://pollack.research.engineering.cornell.edu/
Dr. Ros’ research currently focuses on bioanalytical microfluidics. Her major interests include the development of analytical techniques exploiting micro- and nanoenvironments to develop novel migration phenomena and separation techniques, single cell analysis and tools for nanocrystallography.
Dr. Ros uses her expertise in bioanalytical microfluidics and teams with the STC collaborators in developing novel tools for protein crystallography. She is in particular interested in developing novel microfluidic methodology for size fractionation of protein crystals in the sub-micrometer range and microfluidic devices to reveal the regimes of nanocrystal growth. In the past, Dr. Ros and coworkers have shown a microfluidic sorter based on dielectrophoresis to fractionate photosystem I crystals into size fractions as small as 100 nm. Microfluidic crystallization coupled to numerical simulation also successfully demonstrated the regimes of photosystem I crystal growth. Dr. Ros will support the BioXFEL team with these innovative approaches and further improve microfluidic tools for BioXFEL.
The Ros lab will develop novel microfluidic tools for protein crystallography. A focus lies on tools to fractionate crystals into homogeneous size fractions for improved structure determination. BioXFEL will also be supported with the development of novel microfluidic approaches for sample injection and characterization of nanocrystal growth. http://roslab.chemistry.asu.edu/
Dr. Saldin's research covers all aspects of the use of radiation to study matter, and diffraction and scattering phenomena. His particular interests center around holography, tomography, and x-ray scattering from biomolecules.
Dr. Saldin's contributions to the BioXFEL relate to how proteins and viruses perform their functions in a living organism in an aqueous solution. Knowing that the standard method of structure determination by X-rays require the formation of crystals, it is unclear that the structures of biomolecules in crystals are always the same as their structures in living organisms. This is the motivation for his lab's work on structure determination of biomolecules and viruses in aqueous solution. When used with prior X-ray sources, the technique of small angle X-ray scattering (SAXS) is able to recover the shapes of biomolecules in solution by analyzing the radial distribution of scattered intensities. The shortness of XFEL pulses enables much more detailed information about biomolecules and viruses to be gleaned from such experiments by analyzing also the angular correlations of the intensities of temporally frozen orientations. When averaged over a large number of XFEL diffraction patterns, the angular correlations can yield more detailed information about the structures of biomolecules, independent of the particle orientations. Such methods are ideal to maximize the measured signals from the particles under study as they enable a solution to be used which is sufficiently concentrated, as a higher signal-to-noise ratio is possible if the beam scatters off multiple particles. By computer simulations the Saldin's lab has shown how such a method can reconstruct the two main classes of viruses, namely the icosahedral and helical. https://people.uwm.edu/saldin-research-group/
Marius Schmidt is using physical methods to investigate biological molecules. He is concentrating mainly on static and time-resolved macromolecular crystallography to investigate the structure, dynamics and kinetics of proteins. Dr. Schmidt is also developing new, sophisticated software for the analysis of the time-resolved (4-dimensional) X-ray data. Results from crystallography are corroborated by (time or energy resolved) spectroscopic methods.
Dr. Schmidt's lab, as part of the BioXFEL Center, will work on time-resolved studies on photoactive proteins and enzymes at near-atomic resolution. The time-resolution varies between fs to observe the earliest events in laser activated reactions, and ns and longer to investigate enzymatically catalyzed reactions. They will employ time-resolved serial femtosecond (nano) crystallography (TR-SFX), as well as time-resolved X-ray scattering from solution to determine atomic structures of transient intermediates of these reactions. http://pantherfile.uwm.edu/smarius/www/
Dr. Snell's research group uses complementary techniques to extract structural and dynamic information from biological macromolecules. This research includes the development of crystallization methodology and the resulting analysis with an emphasis on high-energy light sources. Other techniques in use include Electron Paramagnetic Resonance and spectroscopy. He is experienced in solution scattering techniques, having organized and taught at both national and international meetings.
Dr. Snell, as part of the BioXFEL Center will focus his research effort in the SAXS-based study of alternate conformations in macromolecular complexes.
The John Spence Group will focus on development of new single-particle sample-delivery systems for viruses and macromolecules, which aim to improve the hit rate from its current value of about 1% in gas-phase injectors, which are free of water background. Secondly, they will continue development of new data analysis methods for both nanocrystals and single particles. These include use expectation maximization for partial reflections and angular correlation methods for single particles. The Spence Group is also heavily involved in improvement of pump-probe time-resolved nanocrystallography instrumentation. Work on the development of slower jets, which require use of much less protein, continues in collaboration with the Weierstall group also at ASU.
[https://live-spence.ws.asu.edu/ The John Spence Lab at Arizona State University]
The Wierstall Group hopes to develop new and improved injector designs for single particle imaging, as well as for nanocrystal experiments. The single particle injector design has to improve on the poor hit rate of existing designs and evaporate the water surrounding the particles completely, but gently enough to not damage the molecules. Work on the nanocrystal injectors has the goal to reduce the sample waste of current injectors, i.e., by using higher viscosity liquids or pulsing the jet. Work will also begin on making injection nozzles more reproducible, by i.e., mass fabrication with lithography techniques. All new designs will be inspired by the experimental demands of the BioXFEL community and will be implemented during beamtimes at XFELs with the BioXFEL Center. http://www.public.asu.edu/~weier/ASU_site/Home.html