Abstract
The analysis of a single-particle imaging (SPI) experiment performed at the AMO beamline at LCLS as part of the SPI initiative is presented here. A workflow for the three-dimensional virus reconstruction of the PR772 bacteriophage from measured single-particle data is developed. It consists of several well defined steps including single-hit diffraction data classification, refined filtering of the classified data, reconstruction of three-dimensional scattered intensity from the experimental diffraction patterns by orientation determination and a final three-dimensional reconstruction of the virus electron density without symmetry constraints. The analysis developed here revealed and quantified nanoscale features of the PR772 virus measured in this experiment, with the obtained resolution better than 10 nm, with a clear indication that the structure was compressed in one direction and, as such, deviates from ideal icosahedral symmetry.
Highlights
Single-particle imaging (SPI) performed using hard X-ray free-electron lasers (XFELs) (Altarelli et al, 2006; Emma et al, 2010; Ishikawa et al, 2012) was proposed more than a decade ago (Neutze et al, 2000; Miao et al, 2001; Gaffney & Chapman, 2007) as a method of determining the structure of individual biological samples from viruses to single molecules in their native environment
XFELs generate pulses with ultra-high brilliance and high spatial coherence on the femtosecond time scale that is a prerequisite for the success of SPI at XFEL sources
The data analysis consisted of multiple steps of classification and filtering; useful single-hit diffraction patterns were defined as those where only a single virus was present in the XFEL beam
Summary
Single-particle imaging (SPI) performed using hard X-ray free-electron lasers (XFELs) (Altarelli et al, 2006; Emma et al, 2010; Ishikawa et al, 2012) was proposed more than a decade ago (Neutze et al, 2000; Miao et al, 2001; Gaffney & Chapman, 2007) as a method of determining the structure of individual biological samples from viruses to single molecules in their native environment. The data analysis consisted of multiple steps of classification and filtering; useful single-hit diffraction patterns were defined as those where only a single virus was present in the XFEL beam. This single-hit class contains the most valuable data for structure determination by SPI.
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