CAREER: Imaging dynamic macromolecules in solution with x-ray lasers

CAREER: Imaging dynamic macromolecules in solution with x-ray lasers CAREER: Imaging dynamic macromolecules in solution with x-ray lasers Biological macromolecular dynamics have always been of central importance to structural biology, because macromolecules undergo dynamic structural changes and interact with their environment in various ways in order to carry out their functions. When X-Ray Free-Electron Laser (XFEL) facilities began operating in 2009, they brought unprecedented capabilities for atomic-resolution imaging through x-ray scattering on timescales down to a few femtoseconds (10-15 seconds). XFELs generate femtosecond x-ray pulses of extreme intensity that circumvent the fundamental problem of x-ray radiation damage by outrunning atomic motion, which enables room temperature measurements. This capability has become increasingly important to structural biology, as indicated by more than 134 atomistic protein models that have been determined thus far. Many of these structures are time-resolved and comprise molecular movies that are created by exciting macromolecules through various means. The insights gained in this way lead to a better understanding of how macromolecular dynamics relate to life and disease. For example, it was recently shown how a molecular movie can guide the modification of a light-activated protein in order to improve its photo-switching efficiency for biotechnology applications, such as optical super-resolution imaging of whole cells. This proposal aims to develop XFEL-based methods for the study of macromolecules in solution, and to apply these methods to the study of light-activated proteins in order to better understand the fundamental biophysics that underlie their dynamical functions. We aim to move beyond the serial femtosecond crystallography technique that has dominated XFEL-based structural studies so far. This will avoid the need to grow crystals, which is problematic for cases in which crystal packing restricts dynamics, or when crystallization conditions cannot be identified. The proposed developments utilize the unique properties of XFELs in order to produce signals that increase the information content of x-ray scattering patterns to the point that model-free computational images may be formed with little or no prior structural information, without freezing, and with extensions to dynamic studies. We explore the use of femtosecond x-ray inelastic temporal correlations as well as elastic spatial correlations in order to enable imaging of molecules, as well as chemically selective atomic substructures. Exploration of the resolution limits of these novel methods is expected to be important in the coming age of megahertz XFELs that will enable rapid atomic structure determination (structures within seconds), in which crystallization will be the central bottleneck that limits our ability to conduct, for example, combinatorial studies of enzyme reaction dynamics upon rapid mixing with a range of reactants and biochemical conditions. Intellectual merit: This proposal will provide new insights into the fundamental limits of imaging methods based on XFEL intensity-correlation measurements, including x-ray quantum optics. We will come to an understanding of how various experimental factors play into resolution limits. Insights into macromolecular dynamics on picosecond timescales will result, which will challenge our basic understanding of the dynamics of light-activated proteins, and their interactions with surrounding solvent or membrane environments, on timescales down to a few picoseconds. Broader Impacts: The development of a new solution-based method for imaging macromolecular dynamics at angstrom resolutions would be of immense importance to the entire field of structural biology, including the study and treatment of disease, and the development of biotechnologies such as those that harvest and store solar energy. Giant x-ray lasers and life-giving macromolecular machines alos capture the imagination of the broader public and future scientists. The education program interleaves XFEL imaging science with public outreach, includes course development at Arizona State University, and an outreach program tailored to first-generation and low-income community college students. Open-source software and interactive optical diffraction displays will help teach the concepts of diffractive imaging to students and the public. PhD students and undergraduate students will be provided with training, mentorship, career guidance, and will be immersed in a network of other students who also work in the field of XFEL science. The proposed research is highly multidisciplinary and touches upon aspects of microfluidics, computational imaging, x-ray quantum optics, biophysics, mathematics and computer science.