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ABSTRACT Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of

the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure

from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes. Access through

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_PLoS One_ 8, e74113 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We acknowledge valuable discussions with H. Chapman, E. Lattman, J.

Spence and I. Vartaniants. The research conducted at University of Wisconsin–Milwaukee was supported by the US Department of Energy, Office of Science, Basic Energy Sciences, under contract

DE-SC0002164 (A.O., algorithm design and development) and by the US National Science Foundation (NSF) under contract STC 1231306 (A.O., numerical trial models and data analysis; M.S., data

analysis) and under contract number 1551489 (A.O., underlying analytical models). The research at Arizona State University was supported by the NSF under contract STC 1231306 (B.G.H.). Use

of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under

contract DE-AC02-76SF00515. AUTHOR INFORMATION Author notes * Ahmad Hosseinizadeh, Ghoncheh Mashayekhi, Jeremy Copperman, Peter Schwander and Abbas Ourmazd: These authors contributed equally

to this work. AUTHORS AND AFFILIATIONS * Department of Physics, University of Wisconsin–Milwaukee, Milwaukee, Wisconsin, USA Ahmad Hosseinizadeh, Ghoncheh Mashayekhi, Jeremy Copperman, 

Peter Schwander, Ali Dashti, Reyhaneh Sepehr, Russell Fung, Marius Schmidt & Abbas Ourmazd * Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California,

USA Chun Hong Yoon & Andrew Aquila * Biodesign Center for Immunotherapy, Vaccines and Virotherapy, Arizona State University, Tempe, Arizona, USA Brenda G Hogue * Biodesign Center for

Applied Structural Discovery, Arizona State University, Tempe, Arizona, USA Brenda G Hogue * School of Life Sciences, Arizona State University, Tempe, Arizona, USA Brenda G Hogue *

Brookhaven National Laboratory, Upton, New York, USA Garth J Williams Authors * Ahmad Hosseinizadeh View author publications You can also search for this author inPubMed Google Scholar *

Ghoncheh Mashayekhi View author publications You can also search for this author inPubMed Google Scholar * Jeremy Copperman View author publications You can also search for this author

inPubMed Google Scholar * Peter Schwander View author publications You can also search for this author inPubMed Google Scholar * Ali Dashti View author publications You can also search for

this author inPubMed Google Scholar * Reyhaneh Sepehr View author publications You can also search for this author inPubMed Google Scholar * Russell Fung View author publications You can

also search for this author inPubMed Google Scholar * Marius Schmidt View author publications You can also search for this author inPubMed Google Scholar * Chun Hong Yoon View author

publications You can also search for this author inPubMed Google Scholar * Brenda G Hogue View author publications You can also search for this author inPubMed Google Scholar * Garth J

Williams View author publications You can also search for this author inPubMed Google Scholar * Andrew Aquila View author publications You can also search for this author inPubMed Google

Scholar * Abbas Ourmazd View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.H.: algorithmic design, data preprocessing, single-particle hit

finding, orientation and structural recovery, preparation of paper. G.M.: conformational analysis, preparation of paper. J.C.: conformational analysis and validation, preparation of paper.

P.S.: experimental and algorithmic design, code development, data preprocessing, data analysis, experiments at LCLS, preparation of paper. A.D., R.S. and R.F.: data-analytical contributions.

M.S.: analysis of results, preparation of paper. C.H.Y.: experimental design, data collection. B.G.H.: sample selection, preparation and characterization, experiments at LCLS, preparation

of paper. G.J.W.: planning and execution of experiment and discussions of data and analysis. A.A.: experimental design, data collection, preparation of paper. A.O.: experimental and

algorithmic design, data analysis and interpretation, preparation of paper. CORRESPONDING AUTHOR Correspondence to Abbas Ourmazd. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 2D SNAPSHOTS OBTAINED BY DIRECT PHASING OF INDIVIDUAL DIFFRACTION SNAPSHOTS. Each row shows five

representative snapshots assigned to an extreme value of the conformational parameter τ. Note the reduction in the projected inner density between the two rows. (Rainbow color code, with

dark red corresponding to the highest density.) SUPPLEMENTARY FIGURE 2 RELIABILITY OF THE RECONSTRUCTED DIFFRACTION VOLUME. R-split as a function of the magnitude of the scattering vector

_q_, indicating reliable reconstruction up to the detector-limited resolution of 9 nm. The analysis was performed by random splitting, without substitution, of the 37,550 snapshots into two

disjoint datasets. Source data SUPPLEMENTARY FIGURE 3 SPATIAL RESOLUTION OF 3D DENSITY. Fourier Shell Correlation (FSC) vs. the magnitude of the scattering vector _q_ obtained from splitting

the 37,550 snapshots into two random disjoint subsets. The resolution is limited to 9 nm by the detector geometry. Source data SUPPLEMENTARY FIGURE 4 SPHERICALLY AVERAGED RADIAL DENSITIES

OF 3D STRUCTURES OBTAINED WITHOUT IMPOSING ICOSAHEDRAL SYMMETRY. The conformational coordinate ranges from 0 (black curve) to 1 (red curve). Averages (circles) and standard deviations (error

bars) pertain to twenty 3D iterative phasing calculations using all 37,550 single-particle snapshots. Spline fits are guides to the eye. Source data SUPPLEMENTARY FIGURE 5 A REPRESENTATIVE

EXPERIMENTAL XFEL DIFFRACTION SNAPSHOT OF THE PR772 VIRUS BEFORE AND AFTER PREPROCESSING. (A) A raw snapshot. (B) Same snapshot after background correction. The dark horizontal line in each

snapshot is due to the gap between the two detector panels. Intensity is shown in the rainbow color code, with red corresponding to the highest intensity. SUPPLEMENTARY FIGURE 6 DIFFUSION

MAP MANIFOLDS FORMED BY EXPERIMENTAL XFEL DIFFRACTION SNAPSHOTS OF THE PR772 VIRUS. (A) Manifold obtained by embedding 135,375 preprocessed XFEL snapshots (“hits”). (B) Distribution of

single- and multi-particle snapshots on the same manifold. The general parabolic shape stems from changes in the incident beam intensity intersecting the particles. (C) Manifold of

single-particle snapshots after outlier removal. A total of 37,550 single-particle snapshots were extracted by this analysis. Source data SUPPLEMENTARY FIGURE 7 COMPARISON OF ICOSAHEDRAL

WIGNER D FUNCTIONS WITH DIFFUSION MAP MANIFOLDS OBTAINED FROM SIMULATED AND EXPERIMENTAL DIFFRACTION SNAPSHOTS. (A) Icosahedral Wigner D-functions sampled in 30,000 randomly selected

orientations. (B) DM eigenfunctions obtained from 30,000 simulated, noise-free snapshots of an icosahedral capsid. (C) Manifold from 37,550 simulated snapshots of an icosahedral capsid with

experimental signal-to-noise ratio (SNR) and detector gap. (D) Manifold from 37,550 experimental single-particle PR772 snapshots. (E) Eigenvalue spectrum for an icosahedral capsid simulated

at the experimental SNR. (F) Eigenvalue spectrum for the PR772 virus. Source data SUPPLEMENTARY FIGURE 8 DIFFUSION MAP EIGENVALUE SPECTRA FOR EXPERIMENTAL AND SIMULATED DIFFRACTION SNAPSHOTS

OF PR772 VIRUS. (A) Spectrum for 37,550 single-particle experimental snapshots of PR772 virus. (B) Spectrum resulting from the same number of simulated snapshots of an icosahedral capsid

stretched by 10% along one 5-fold axis. Note the differences between the eigenvalue spectra. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–8

and Supplementary Note (PDF 1339 kb) LIFE SCIENCES REPORTING SUMMARY Life Sciences Reporting Summary (PDF 160 kb) SUPPLEMENTARY DATA Single-particle indices (XLSX 1153 kb) 3D CONFORMATIONAL

MOVIE WITH IMPOSED ICOSAHEDRAL SYMMETRY. Evolution of the 3D structure of the PR772 virus, and the occupancy along the dominant conformational reaction coordinate. These results were

obtained assuming icosahedral symmetry. (MOV 800 kb) 3D CONFORMATIONAL MOVIE WITHOUT IMPOSING ICOSAHEDRAL SYMMETRY. Evolution of the 3D structure of the PR772 virus, and the occupancy along

the conformational reaction coordinate without imposing icosahedral symmetry. Note the protrusion of a tubular structure, and the concentration of the genome toward the tube. (MOV 418 kb)

SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO SUPPLEMENTARY FIG. 3 SOURCE DATA TO SUPPLEMENTARY FIG. 4 SOURCE DATA TO SUPPLEMENTARY FIG. 5 SOURCE DATA TO

SUPPLEMENTARY FIG. 6 SOURCE DATA TO SUPPLEMENTARY FIG. 7 SOURCE DATA TO SUPPLEMENTARY FIG. 8 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Hosseinizadeh, A., Mashayekhi, G., Copperman, J. _et al._ Conformational landscape of a virus by single-particle X-ray scattering. _Nat Methods_ 14, 877–881 (2017).

https://doi.org/10.1038/nmeth.4395 Download citation * Received: 03 February 2017 * Accepted: 05 July 2017 * Published: 14 August 2017 * Issue Date: 01 September 2017 * DOI:

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