Abstract
A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome–capsid interface.
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Data availability
CG packaging trajectories and select all-atom MD trajectories with water molecules removed have been deposited in the Illinois Data Bank under accession code IDB-4930709 (ref. 90). The following structures were used from the PDB: 3KDR, 2FT1 and 1OHG. Source data are provided with this paper.
Code availability
The files needed to setup CG packaging simulations and select all-atom MD simulations have been deposited, along with select analysis scripts, in the Illinois Data Bank under accession code IDB-4930709 (ref. 90). Remaining analysis scripts are available upon reasonable request.
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Acknowledgements
This work was supported by the National Institute of General Medical Sciences grant R01-GM137015, the National Science Foundation grant PHY-1430124 and the Human Frontier Science Project (RGP0047/2020). ARBD development is supported by the National Science Foundation grant OAC-2311550. The supercomputer time was provided through the Leadership Resource allocation MCB20012 on Frontera of the Texas Advanced Computing Center and the XSEDE allocation MCA05S028. We thank J. Johnson for sharing SAXS data for the empty HK97 capsid, and A. Evilevitch, I. Golding, R. Duda and S. Butcher for discussions.
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A.A. conceptualized and supervised the work and performed project administration. A.A., K.C. and C.M. designed the methodology. K.C., C.M. and D.W. performed the investigations and visualizations. A.A., K.C., C.M. and D.W. acquired funding. A.A., K.C., C.M. and D.W. wrote the original draft. A.A., K.C. and C.M. reviewed and edited the article.
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Extended data figures and tables
Extended Data Fig. 1 Structure and fluctuations of empty HK97 capsid.
a, RMSD of the entire capsid from its initial coordinates during all-atom equilibration simulations. For the first 70 ns, parts of the system were subject to restraints, as detailed in Methods and Supplementary Table 2. The image on the right shows one of the 60 asymmetric subunits with residues resolved in the crystal structure shown in blue and modeled in red. b, Arrangement of the asymmetric subunits into an icosahedron capsid (left). Modeled residues are shown red. The right image details the subunit, consisting of seven proteins. c, Distribution of individual proteins’ average RMSD grouped according to the protein location in the asymmetric subunit. The distributions were computed over the last 38 ns of the equilibration trajectory. Colors are defined in b. d, Average per-residue RMSD of an empty capsid from the crystallographic coordinates as a function of the residue number. RMSD of modeled residues is not shown. e, Cartoon representation of resolved regions of proteins 1 (representative for proteins 1 to 5), 6 and 7 colored according to their average RMSD. f, Average RMSF of individual proteins according to their location. The RMSF values were computed over the last 100 ns of the equilibration trajectory. Error bars depict SD over n = 60 copies of the protein. The capsid is shown (right), with individual residues of the proteins colored by their average RMSF. g, Cumulative variance of the principal components (PCs). PC analysis was performed using CoM coordinates of each of the 420 capsid proteins and a representative 150 ns fragment of the free equilibration trajectory. (Top) Fractional cumulative variance as a function of the number of PCs, ordered by eigenvalue from highest to lowest. (Bottom) Same, shown only for the top twenty principal components. h, Projections of the top three PC, shown as vectors drawn from an average structure. Images in (i), (ii) & (iii) show projections of the first, second and third PCs, ordered by eigenvalue from high to low, each shown from four different perspectives. The top three PCs account for about 23, 12 and 6% of the total variance, respectively. Vector magnitudes drawn are based on the normalized eigenvector multiplied by the corresponding eigenvalue and then multiplied by a factor of ten, to facilitate visualization.
Extended Data Fig. 2 Electrostatic properties of empty and packaged capsids.
a, Ion exchange rate during equilibration of the empty (left), “slow” (center) and “slow with twist” (right) packaged capsids. Ion exchange data were collected every 10 ns. The lines shown were obtained from the data using a Savitzky-Golay filter with a 110 ns window. b, Radial profiles of the electrostatic potential averaged over 50 ns (empty; left) or 100 ns (slow and slow with twist; center and right) windows. The electrostatic potential was computed using VMD PMEpot plugin91. c, Image depicting the final result of an algorithm used to select the interior and exterior of the capsid, see Methods for details. d, Total charge inside the protein capsid in the units of proton charge. Traces are shown with (solid lines) and without (dotted lines) inclusion of the protein capsid in the analysis. Water molecules were neglected in the analysis. e, Charge inside a spherical volume centered at the center of the capsid as a function of the sphere’s radius. Data were averaged over the last 100 ns of each simulation. For reference, the density of Cα atoms is shown (blue histogram; right axis). f, Electrostatic dipole moment of an asymmetric subunit projected along the radial axis. At each frame, the moment was averaged over all sixty copies of the asymmetric subunit. For reference, the blue line shows the average dipole moment of the residues resolved in the X-ray structure. g, Theoretical model used to estimate the electrostatic potential across the capsid (see Methods). h, Average potential difference between solvent occupied regions inside and outside the capsid as computed using PMEpot (circles) and from a fit using the theoretical model (dashed lines). In contrast to analysis shown in Fig. 3i, j, the PMEpot averaging was restricted to the solvent region within the capsid. That was done by blurring the mass density of DNA nucleobases via convolution with a 1 nm wide gaussian kernel and then selecting regions where the blurred density was less than 0.05 Da Å−3. The fit was performed by minimizing the MSD between predicted and measured data points, yielding the following values for the parameters of the model: qextra, empty = 248.4 e, qextra, slow = 404.9 e, and εpro = 6.53.
Extended Data Fig. 3 Properties of packaged genome configurations.
a, Sixteen genome configurations obtained by independent coarse-grained packaging simulations performed under the same 55 pN packaging force in the absence of twist (left) and with a 14o-per-10-bp twist imposed by the packaging protocol (right). Each genome configuration has a unique interface between the early- (blue) and late-packaged (red) DNA domains. b, Statistical properties of the packaged DNA configurations. Each plot depicts the distributions of a locally-defined metric for beads assigned in five different radial ranges, each with a 5 nm width, except the innermost group, which includes all beads within 7.5 nm of the capsid center. The distributions are semi-transparent and are provided for each packaged capsid. The mean of each distribution is shown as a solid horizontal line segment. The first three columns of plots characterize the angle between the local tangent at each bead and the spherical basis vectors at the bead’s location. The last three columns depict local curvature, toroidal order27,51 and nematic order. The fast packaged capsids exhibit significant differences according to several of the metrics. For example, the fast packaged DNA located within 7.5 nm of the capsid center points away from the portal by 5o on average, whereas the slow packaged DNA points towards and away from the portal with roughly equal likelihood. Compared to the slow packaged genomes, the fast packaged DNA has a slightly higher average curvature, lower nematic order, and, especially when twist was not imposed, greater toroidal order.
Extended Data Fig. 4 Structural features of packaged capsids.
a, Trajectory-average distance from the center of the capsid to the CoM of each capsid protein versus the corresponding crystal structure value. For each protein location, the symbol shows the value averaged over the 60 copies of the protein, whereas the histogram (top axis) shows the distribution among the protein copies. Data are shown from the last 50 ns of each trajectory, sampling coordinates every 0.192 ns. b, Average per-residue RMSD of the “slow” and “slow with twist” capsids from the crystallographic coordinates as a function of the residue number. RMSD of modeled residues is not shown. The averaging was done over the last 105 ns of each trajectory, sampling coordinates every 0.192 ns. The proteins at locations 1 to 5 exhibit consistent patterns (left), and the average of these data is depicted on the right (black). c, Average per-residue displacement of the packaged capsid with respect to the empty capsid. Deviation of modeled residues is not shown. The averaging was done over the last 40 ns of each trajectory, sampling coordinates every 48 ps. The mean and standard deviation over protein locations 1 to 5 are shown in black and grey, respectively.
Extended Data Fig. 5 Supplementary analysis of the all-atom MD trajectories.
a, Location of water passages (blue molecular surface) within the capsid visualized as an isosurface (5.26 molecules nm−3) of water density, averaged over the 60 icosahedron subunits, for the empty (left) and packaged (right) capsids. The analysis was performed using the last 2 ns of the each MD trajectory. The data in the left image are the same as in Fig. 1i. The ratio of the volume occupied by water (slow to empty) is 0.68. Less prominent differences in the water passages are observed inside the region confined by the yellow semi-transparent curve. Prominent differences are observed outside that region, where adjacent asymmetric subunits meet. Thus, expansion of the capsid caused by the packaged DNA leads to reduction of the gaps between the adjacent protein subunits. b, Diffusivity of DNA helices plotted as a function of their radial distance from the capsid’s center. Error bars reflect standard deviation across all points within each radial bin. Data shown in the upper panels were obtained in the reference frame of the capsid whereas those in the lower panels were obtained in the local helical reference frame. The insets schematically illustrate the motion characterized by the corresponding diffusion coefficients. c, Radial distribution of ion diffusivity (left axis). Symbols correspond to the simulation models: empty circles for “empty”, yellow diamonds for “slow with twist” and green squares for “slow”, respectively. The radially averaged protein density (right axis) is shown for the packaged (filled distribution) and empty (dashed line) simulation trajectory.
Extended Data Fig. 6 TEM-like analysis of packaged HK97 genomes.
a, TEM images of HK97 viral particles. b, TEM-like images of computationally packaged HK97 genomes. To make the TEM-like images, DNA mass density obtained from the coarse-grained packaging simulations was projected along several axes for several packaged genome configurations. The capsid density was not included in the analysis. Image in a reproduced from ref. 17.
Extended Data Fig. 7 Properties of packaged genomes according to all-atom MD simulations.
a, Simulated profiles of DNA density for the four microsecond-long all-atom MD trajectories. The simulated profiles were computed by averaging 4 × 4 nm2 centered sections connecting the ten pairs of opposite faces (left) or the six pairs of opposite vertices (right) and by averaging over the last 500 ns of the respective trajectories. The conformations resulting from the slow packaging simulations show greater DNA ordering (more visible layers) compared to the conformations resulting from fast packaging simulations. Lower but persistent order is observed along the vertices’ symmetry axes. The configurations sampled by the simulations of the “slow” packaged particle show a higher ordering of the DNA near vertices compared to other simulations. The variations observed in individual structures may arise from the inherent stochasticity associated with the process of packaging. b, Fraction of base pairs broken in the DNA genome during the equilibration simulations of the six packaged particles. A base pair is considered intact if the H1 or N1 atom of a purine is within 2.5 Å of the N3 or H3 atoms of a pyrimidine, and the angle formed by the N1-H1-N3 or N1-H3-N3 atoms is greater than 115 degrees. c, Fraction of base pairs broken within three internal radial bins, analyzed every 0.96 ns of the “slow” (solid lines) and “slow with twist” (dotted lines) trajectories. d, Fraction of broken basepairs characterized according to their conformation (frayed, mis-stacked and over bent), analyzed over the last 50 ns of the “slow” trajectory. Exterior (Ext.) refers to DNA base pairs that have at least one non-hydrogen atom within 20 Å of the protein non-hydrogen atom, and interior (Int.) refers to all other base pairs. Error bars reflect standard deviation across all points within each radial bin. e, Violin plots of the angle between the local axis of a 10 bp DNA fragment and a radial vector as a function of the radial distance to the capsid center. The CoM of each 10 bp fragment was recorded for the last 288 ns of the equilibration trajectory, sampled every 0.48 ns. As expected, helices tend to align transverse to the radial vector, as one moves from the capsid center to the outermost layer (blue). A relatively higher bimodality is observed when packaging is performed at higher force in the bins next to the outermost layer, i.e. the two bins shown in green.
Extended Data Fig. 8 Base pair-level characterization of the all-atom genome structures.
The analysis was performed by first writing down separate coordinate files for every 150 bp of the genome for the last 5 ns of each all-atom trajectory every 48 ps. Each individual coordinate file was then analyzed using the Curves+ package86. The base-pair level properties were then averaged according to the base pairs’ radial distance from the capsid center and normalized with respect to the number of base pairs within each radial bin and error bars depict s.e.m. over n = 104 consecutive segments of the trajectory. Colors indicate the different packaged models. Dashed line depicts mean deviation for two DNA duplexes having a random sequence of 28 bp in a 0.1 mol kg−1 KCl solution, each simulated for 90 ns in the NPT ensemble. For both systems, base-pair parameters were averaged separately for last two 40 ns intervals, giving four independent samples for computing the standard error. Schematic images adapted from ref. 59, Springer Nature.
Extended Data Fig. 9 Topological defects in the structure of DNA genome near the edges of the capsid.
a, Arrangement of the DNA molecules (blue) in the outermost layer of the genome at the end of the all-atom MD equilibration of the packaged capsid (slow trajectory). Proteins forming the capsid edges are shown in pink; the rest of the assembly is not shown for clarity. b, Same as in the previous panel, showing only the DNA helices located within 20 Å of the capsid edges. c, For each capsid edge (pink) the DNA helices from the previous panel are separately shown, viewed from the inside to outside.
Extended Data Fig. 10 Multi-resolution model of DNA–DNA and DNA–protein interactions.
a, Explicit solvent all-atom MD simulations of internal pressure in a DNA array50. Color indicates bulk electrolyte molarity: 20 mM Mg2+/200 mM Na+ (cyan), 250 mM Na+ (orange), and 2 mM Sm4+/200 mM Na+ (red). Using CUFIX corrections to non-bonded interactions63 was essential to achieve quantitative agreement with experiment. b, CG simulations of DNA array pressure at multiple resolutions. The internal pressure matches experimental values regardless of the resolution of the model. The CG simulations were performed using the mrDNA model77. c, Calibration of DNA–protein interactions. In each simulation, a DNA molecule was pushed against a flat cross-section of the viral capsid by an external force corresponding to a 20 bar pressure. The grid-based representation of the protein capsid was tuned to match the average DNA–protein distance seen in the all-atom simulation. Image in a adapted from ref. 50, Oxford Univ. Press.
Supplementary information
Supplementary Information
Supplementary Tables 1–9 and custom patch for crosslinking Lys169 and Asn356 residues.
Supplementary Video 1
All-atom equilibration of an empty HK97 capsid (left) spanning 0.746 microsecond and of a fully packaged capsid (right) spanning 1 microsecond. For clarity, the solvent is not shown. Capsid proteins comprising vertices are colored in light pink, faces in light green and edges in light purple. To make the movie, instantaneous configurations were sampled every 2.88 ns, smoothed with a running window of three consecutive frames, and recorded at 25 frames per second.
Supplementary Video 2
Top three modes (left to right, descending) obtained by performing principal component analysis of the empty capsid equilibration trajectory. Spheres represent the centers of mass of the 420 proteins comprising the capsid, colored by their types, with vertices in pink, faces in green and edges in yellow. The principal component analysis was performed using center-of-mass coordinates of each of the 420 capsid proteins and a representative 150 ns fragment of the free equilibration trajectory, with principal component modes scaled by an amplitude of 2.5 nm.
Supplementary Video 3
Illustration of the multiresolution model used to package the 39,732 base pairs of the HK97 genome. The DNA is colored blue-to-red from first-to-last base pair to be packaged. DNA packaging was produced by applying a local force to the DNA beads located within the capsid’s portal, shown in the cutaway view of the grid potential representing the capsid. In some simulations, additional torque was applied to package DNA with a predetermined degree of twist. The animation also depicts the mapping procedure used to construct an all-atom model of the genome, its merger with the solvent and the all-atom model of the capsid, the restrained relaxation of the combined system followed by the unrestrained equilibration
Supplementary Video 4
Coarse-grained simulations of genome packaging. The top eight models were simulated without a twisting motion; the bottom eight had a local torque applied in the portal region to rotate the DNA right-handed by 14 degrees per one turn of the packaged DNA. DNA is colored in order of entry into the capsid, blue to red. The packaging process spans roughly 1 millisecond of simulation time.
Supplementary Video 5
Spontaneous ejection of the packaged genome through the capsid portal. In 22 milliseconds, approximately 75% of the genome is ejected out. DNA is colored in order of entry into the capsid, with the first beads packaged shown in blue and the last in red.
Supplementary Video 6
Switchback loop formation during packaging simulations. Animation depicts the formation of a switchback loop defined to be a region of DNA with high curvature flanked by two arms that remain within 6 nm. Three switchback loops are formed (in cyan, green, and purple) during this 70-microsecond excerpt of a “slow” packaging trajectory. The time-dependent local bending energy of the DNA averaged over a 1.76 microsecond window is mapped from 0.01-1.5 kcal/mol to a red, white, and blue color gradient. The capsid is cut away during the final 15 microseconds of the excerpt to reveal that some loops protrude deeply into the interior of the genome. The movie is shown at 25 frames per second, with each frame representing 160 ns. For clarity, the coordinates of the DNA were smoothed using a running window over every two consecutive frames.
Supplementary Video 7
All-atom simulation trajectory of a fully packaged HK97 particle (“twist” packaged model) spanning 1 microsecond. A quadrant of the protein capsid (shades of blue) is removed to show the packaged DNA genome. The two DNA strands are colored in shades of red. Ions are shown as colored spheres with sodium shown in yellow, chloride in blue and magnesium in green. For clarity, only a fraction of all ions present in the system is shown. To make the movie, instantaneous configurations were sampled every 2.88 ns, smoothed with a running window of two consecutive frames and recorded at 25 frames per second.
Supplementary Video 8
Visualization of the electrostatic potential map of an empty and packaged capsids at equilibrium. The electrostatic potential was averaged over the last 50 ns of the empty capsid equilibration trajectory or over the last 100 ns of the packaged capsid equilibration trajectory. In the movie, subsequent slices in the xy-plane are progressively shown, with the z-coordinate given on the bottom right in blue.
Supplementary Video 9
Final configurations of the packaged DNA after 1 microsecond equilibration of the corresponding all-atom models. The models derived from the “slow” packaged simulation is shown on the left and the one packaged “slow with twist” is shown on the right. The DNA is colored blue to red according to the radial distance from the center. The color scale ranges uniformly from 250 to 290 angstroms from the capsid center. Red regions protrude into the vertices of the capsid, while blue regions are located near the edges of the capsid. For clarity, the protein capsid, the solvent, and the inner layers of the packaged DNA are not shown. During the movie, the rotation pauses at all twelve vertices of the capsid.
Supplementary Video 10
Schematics of the protocol used to measure internal pressure from an all-atom simulation. At different instances of the simulation trajectory, where pressure is measured, the DNA is replaced with bulk-like solvent and the capsid is restrained with harmonic forces. The force required to restrain the capsid in its expanded configuration reports on the total internal pressure.
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Coshic, K., Maffeo, C., Winogradoff, D. et al. The structure and physical properties of a packaged bacteriophage particle. Nature 627, 905–914 (2024). https://doi.org/10.1038/s41586-024-07150-4
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DOI: https://doi.org/10.1038/s41586-024-07150-4
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