The long-term goal of this project is to determine the three-dimensional structure of viruses at atomic resolution. An accurate knowledge of viral structure is necessary to understand the mechanism of infection at the molecular level. Most enveloped viruses are not amenable to crystallization and X-ray diffraction for structural studies. Having a rapid method for structure determination of viruses, which does not require crystallization, would be a transformative concept for structural biology. Using the high intensity X-ray pulses generated at the world's first X-ray laser, the Linac Coherent Light Source (LCLS) at Stanford, diffraction patterns from aerosolized single virus particles were obtained with previous NSF funding. Due to the short X-ray pulse duration of 10-100 femtosecond, each virus particle generates a diffraction pattern before radiation damage destroys the particle. Two dimensional images of the virus particles could be reconstructed from these patterns. This research follows up on these first preliminary results and expands the method to enveloped viruses, which need to be hydrated in order to preserve their structure. The viruses are sprayed in a liquid jet in vacuum across the X-ray beam using an injector developed at ASU with previous NSF funding. The aim is to improve the resolution of the method by initially working at an X-ray energy, where the contrast between virus and water is maximized. Experiments at higher X-ray energy will follow with the goal to achieve sub nanometer resolution with single virus particles. The patterns from many particle orientations will be classified, sorted, merged and phased to form a three-dimensional image of the virus.

Broader Impact: Outreach activities under this proposal include a partnership with Arizona State University's Science is Fun program. Under this project, Science is Fun will develop a physics based science demonstration and lesson geared towards students in kindergarten through eighth grade. This project will be developed according to national and state educational standards. In the Science is Fun program, trained interns visit K-12 classrooms, facilitating science demonstrations and hands on science activities for students and teachers in 21 school districts across the metro Phoenix area. In addition, the current on campus lab tours will continue. Students in middle school through high school who visit the Arizona State University campus will have the opportunity to tour working labs. State of the Art imaging equipment will be made available for school groups as part of this proposal.

The results of the project will form the basis for a new generation of instrumentation that may rapidly solve virus structures, which are difficult to crystallize. This interdisciplinary research will bring together elements of biochemistry, molecular biology, fluid dynamics, coherent X-ray imaging, classification and phasing algorithm development and in doing so will promote research and learning in integrated science areas that will greatly benefit its participating students and postdoctoral associates.

Project Report

The major goal of the project is to develop the use of X-ray free electron lasers (XFELs) for virus structural studies. The X-ray free electron laser is a new tool that allows damage free structure determination of single bio-particles like viruses. The objective is to study/analyze viruses at different X-ray energies with different injectors, with the ultimate goal to produce 3D and eventually sub-nanometer resolution structures of virus particles. This provides the opportunity to gain structural insight about viruses and their proteins, which is significantly and broadly important for the field of biology. New methods of sample injection into vacuum have been developed and applied succesfully at the Linac Coherent Light Source, the first XFEL in the world at Stanford. Single viruses and small virus crystals have been injected into the LCLS beam and analyzed. The Sindbis virus was chosen as one of our major targets for single particle experiments. In addition, chlorella virus that grows in algae, bacteriophage T4, norovirus capsids, cowpea mosaic virus (CPMV) capsids, adenovirus, and echovirus were analyzed either in the form of nanocrystals or as whole viruses. We also analyzed gold-core palladium or copper shelled nanoparticles as structural mimics of virus particles. Methods where developed for the crystallization of CPMV virus capsides and Sindbis viruses. Several beamtimes at the LCLS resulted in a large amount of data that is still being analyzed. The liquid injector developed at ASU is currently the only injector design which allows completely hydrated injection of proteins, nanocrystals and viruses into the X-ray beam of an X-ray free electron laser while at the same time guaranteeing a 100% hit rate (every X-ray pulse hits the jet), since it produces a micron or submicron liquid jet embedding the bioparticles. Our experiments have shown that single virus imaging is possible, although so far at low resolution. The successful virus crystallization trials may enable the use of femtosecond crystallography methods with viruses. A new injector which generates a microscopic stream of high viscosity liquid containing sample crystals has been developed. This injector allows a drastic reduction of the amount of precious sample necessary for structure determination compared with the liquid injector mentioned above. Development of the high viscosity liquid injection nozzle may revolutionize membrane protein crystallography, especially for human membrane proteins, which are usually grown in Lipidic Cubic Phase, a highly viscous medium.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Michele McGuirl
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Arizona State University
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