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. 2005 Feb 22;102(8):2772-7.
doi: 10.1073/pnas.0409825102. Epub 2005 Feb 7.

Cryo-electron tomography of vaccinia virus

Marek Cyrklaff  1 , Cristina Risco, Jose Jesús Fernández, Maria Victoria Jiménez, Mariano Estéban, Wolfgang Baumeister, José L Carrascosa
Affiliations

Cryo-electron tomography of vaccinia virus

Marek Cyrklaff et al. Proc Natl Acad Sci U S A. .

Abstract

The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4-6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of approximately 360 x 270 x 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of approximately 18-19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.

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Figures

Fig. 1.
Fig. 1.
Isolated VV particles (IMV) preserved by rapid freezing and viewed by cryo-EM. (A) Cryo-electron micrograph. A typical projection image comprising several particles at different orientations is shown. (B) An x–y section through the tomographic reconstruction of the same area in A. (C) One of the particles depicted in four subsequent x–y sections; each section is 24 nm thick and 24 nm apart from each other (no denoising procedure was applied). (Scale bar: 200 nm.)
Fig. 2.
Fig. 2.
Volumetric representation of the reconstructed viral particles. (A and B) Surface rendering highlighting the outer shape and size of the virion. Two orthogonal views along perpendicular axes are shown. (C and D) Translucent representation of the reconstructed virion showing the complex internal structure of the core. The dumbbell-shaped core as seen in one orientation (C) evolves to a morphology that closely follows the outer membrane (D). x, y, and z axes are shown to facilitate identification of the spatial orientation of the respective views.
Fig. 3.
Fig. 3.
General overview of the virus in a translucent model. (A) Section through a denoised and surface-rendered virion showing all of the virus structural elements described in the text. The boxed area is enlarged in B. (B) Schematic view of major domains: ruffles (large black arrows) on the outer membrane (OM), lateral bodies (LB), repeating units (small black arrows) in the palisade (PL), and core membrane (CM). The numbers indicate the relative density values of the corresponding regions marked by the arrows.
Fig. 4.
Fig. 4.
Outer membrane and lateral bodies; denoised and surface-rendered views of the outer membrane fragments (blue) are shown. (A and B) Outer, rough aspect (A) and inner, smooth surface (B). (C and D) Lateral bodies (yellow) attached to the inner side of the outer membrane.
Fig. 5.
Fig. 5.
The spikes and palisade layer at the outer surface of the virus core. (A) An x–y section through the reconstruction cutting through the front view of the palisade layer. (B) Small area containing several spikes' crystallites in top view that, after averaging and symmetrizing, generated the reference for a crosscorrelation search, together with the corresponding diffraction pattern. (C) A top view through the correlation map showing distribution of the spikes and small crystallites. The correlation peaks are bright (high crosscorrelation values) and correspond to the electron densities of spikes seen as black in A and B.
Fig. 6.
Fig. 6.
Pores in the inner membrane and core morphology. (A) Section through a representative denoised tomogram that cuts through the pore in the inner membrane (arrows); the box encircles the entire structure. (B and C) Surface-rendered volumes of the pores along the lumen axes (circles), as viewed from outside and inside the virus core, respectively. (D) A side section of the pore (circle) as visualized in the surface-rendered tomogram. (E and F) Different sections of a 3D core reconstructed area. The sectioned surfaces have been shadowed to highlight the features of the palisade and the membrane (arrowheads), as well as the threaded fibers (white arrows in A and outlined in B). (Scale bars: 50 nm.)
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