1. Volume 22, Issue 6, Pages 843-850 (June 2006)
Native Hepatitis B Virions and Capsids Visualized by Electron Cryomicroscopy 
Kelly A. Dryden, Stefan F. Wieland, Christina Whitten-Bauer, John L. Gerin, Francis V. Chisari, Mark Yeager 
Molecular Cell 
Volume 22, Issue 6, Pages (June 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Isolation of Native HBV Capsids and Analysis by Cryo-EM and 3D Image Processing
(A) Images of negatively stained fractions from the cesium chloride gradient.
(B) Southern blot of fractions probed for HBV DNA. Particles negative for HBV DNA (green outline) were in great excess and primarily represented capsids from the nucleus.
(C) There is close similarity between the 16 and 14 Å resolution 3D maps for capsids that are HBV DNA positive (blue outline) and negative (green outline), respectively (2-fold surface-shaded view [top], central cross-section view [middle], and surface view of the radii within the capsid [bottom]). The native particles encapsidate a dodecahedral cage of density ascribed to ∼1 kb of ssDNA, which is closely associated with the inner wall of the capsid.
(D) Radial density plot of the two structures. The HBV-positive particles (blue) have slightly more density at low radii (<80 Å) than the HBV-negative particles (green). Bars, 500 Å.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Two Sizes of Enveloped HBV Virions and Capsid Particles
(A) Representative cryo-EM images and their averages in the right-hand column. Larger (T=4) and smaller (T=3) sized particles were detected for both the virions and capsids. First row (gold), larger T=4 virions; second row (red), smaller T=3 virions; third row (blue), larger T=4 capsids; and fourth row (purple), smaller T=3 capsids. Note the obvious surface projections on the virions (arrows).
(B) Radial density plots of the corresponding spherically averaged 3D reconstructions for each particle type show superposition of the capsid density in the virion and capsid particles.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Image Analysis of Radii Spanning the Icosahedral Capsid and Virion Envelope for T=4 and T=3 Virions
For each panel, the maps on the left display surface and interior views of the starting model (top) and the final map (bottom), rotated 15° off the 2-fold axis. The maps at the right in each panel are 2-fold views of the entire final map (top) and cropped at the indicated radius (bottom). Image processing was restricted to capsid radii spanning 90–170 Å for T=4 virions (A) and radii spanning 90–155 Å for T=3 virions (B). In both cases, the capsid radii are in blue and the envelope radii are in gray in the cut-away views. The interior of the final maps display icosahedral cores that closely resemble authentic T=4 or T=3 capsids (Figure 1C). In contrast, the density of the envelope was relatively featureless. When image processing was restricted to the envelope radii 170–250 Å (gold) for the T=4 virions (C) and radii 155–240 Å (gold) for the T=3 virions (D), the final maps displayed surface projections spaced ∼60 Å apart. The interior radii (gray) that corresponded to the capsid shell became smooth or less well defined.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Image Analysis of HBV Envelope Surface Projections
(A) Maps with imposed icosahedral symmetry arising from the same virion data set but different starting models yielded variable yet comparably spaced surface features. Surface-shaded views of three representative maps (out of eight) viewed along the 2-fold (2F), 5-fold (5F), and 3-fold (3F) symmetry axes. Map 1, the same as shown in Figure 3C; map 2, result from the final map from refinement of density within radii spanning the capsid used as the starting model (Figure 3A); and map 3, result from a starting model generated by applying random orientations to the particles.
(B) Despite completely different starting models, each map displayed similar size and packing of the surface projections but had more poorly defined density at varying icosahedral symmetry positions. This pattern may arise if the projections have ordered packing with random insertion of pentamers. Packing of the surface projections is emphasized by the overlay of yellow dots. The red triangle has 60 Å edges and shows the average spacing of the projections. Five-fold and 3-fold axes are indicated by black pentagons and triangles, respectively.
(C) Analysis of en face images of HBV. Central, en face views of the T=4 capsid X-ray structure (a), cytoplasmic capsids (b), and virions (c, d, and e) were classified by factor analysis. For all the particle types, class averages (left column) displayed discrete densities, which gave rise to sampled reflections in the Fourier transforms (FFT) (middle column). Circularly averaged, 1D profiles of the FFTs (right column) displayed broad peaks. The capsid X-ray structure (a) and the cytoplasmic capsids (b) displayed very similar profiles. A peak at 1/43 Å corresponded to the separation between adjacent spikes and a smaller peak at 1/80 Å was interpreted as diffraction between the second nearest neighbor of spikes. The virion images yielded three classes with different profiles. Class I showed a peak at 1/45 Å, arising from diffraction between the capsid spikes (as in a and b). Class II showed a peak at 1/60 Å, interpreted as diffraction between the envelope projections. Class III showed two peaks at 1/42 Å and 1/61 Å, representing the combination of the capsid and envelope peaks seen in c and d.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Model and Cartoon of HBV Virions
(A) Cut away views of a composite model of the HBV virion comprised of a T=4 icosahedral capsid with 120 spikes and an outer envelope with protein projections spaced ∼60 Å apart. Views are cross-sections (left), and two cut aways.
(B) X-ray crystal structure of recombinant capsid docked into the cryo-EM density map of the virion capsid (left). The tips of the core spikes are in close apposition but do not penetrate the envelope. Additional details and cartoon of interpretation (right). The surface protein projections are ascribed to HBsAg and are designated as large (L), medium (M), and small (S) according to the three start codons of the HBsAg ORF. Residues in the capsid tip are colored according to charge and hydrophobicity (negative, red; positive, blue; hydrophobic, gold; and hydrophilic, gray) and show a mixed distribution of charge. Residues denoted by green spheres have been implicated in viral envelopment via interaction with the L protein (Ponsel and Bruss, 2003). HBcAgs (yellow) modeled as S box with M and L loops. Note that ∼50% of the L molecules have an interior loop that is predicted to be disordered, which interacts with specific residues in HBcAg (green spheres). Bar, 100 Å.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions