Figure 4.
Figure 4. Tomography of negatively stained VSV incubated at pH 7.5 and 5.5, and comparison of the structure of G with the corresponding x-ray crystallography model. (A and B) Three sections of the tomograms are shown (extracted from Videos 1 and 3); one at the level of the G layer (left), one at the level of the nucleocapsid (middle) and one passing through the center of the particle (right). The tilted series used to calculate the tomograms were recorded on negatively stained samples. (A) At pH 7.5, VSV is bullet shaped, with a central cavity. In some areas, the G layer contains trimeric entities (arrows in the enlargement). As the stain does not penetrate the viral particle, the nucleocapsids are not visible. (B) At pH 5.5, G shows trimeric structures that form quasi-helical arrays (left). The nucleocapsid is now visible (middle and right). In the center of the particle, a twisted material occupies the central cavity. (C) Enlargement of the central section (indicated by the arrow in the right frame in B) showing the organization of the particle beneath the membrane. The characteristic bilobed shape of N is visible and an additional domain is seen that may be attributed to M (Ge et al., 2010). (D and E) Volumes at the surface of the particle, extracted from the tomograms, revealing the presence of trimeric entities to which x-ray models of the pre-fusion (D) and post-fusion (E) structures can be manually fitted. In each case, two models (in blue and red) are displayed. (F and G) For a more quantitative fit, four trimers were isolated from the reconstructions and averaged. The x-ray models were fitted to the resulting averaged 3D reconstructions with UROX (Siebert and Navaza, unpublished program), a more user-friendly version of URO (Navaza et al., 2002). (F) For viruses incubated at pH 7.5, two fits are shown. For the fit displayed above and to the left, the crystallographic trimer, with its domains shown in color, was directly fitted in the electron microscopy reconstruction. The second fit (below and to the right) was performed with a monomer, imposing C3 symmetry. The better fit obtained with this model suggests that the trimer of G at the surface of the particle is slightly different from that in the crystalline structure. (G) At pH 5.5, the crystalline post-fusion model of G fits the tomographic model well.

Tomography of negatively stained VSV incubated at pH 7.5 and 5.5, and comparison of the structure of G with the corresponding x-ray crystallography model. (A and B) Three sections of the tomograms are shown (extracted from Videos 1 and 3); one at the level of the G layer (left), one at the level of the nucleocapsid (middle) and one passing through the center of the particle (right). The tilted series used to calculate the tomograms were recorded on negatively stained samples. (A) At pH 7.5, VSV is bullet shaped, with a central cavity. In some areas, the G layer contains trimeric entities (arrows in the enlargement). As the stain does not penetrate the viral particle, the nucleocapsids are not visible. (B) At pH 5.5, G shows trimeric structures that form quasi-helical arrays (left). The nucleocapsid is now visible (middle and right). In the center of the particle, a twisted material occupies the central cavity. (C) Enlargement of the central section (indicated by the arrow in the right frame in B) showing the organization of the particle beneath the membrane. The characteristic bilobed shape of N is visible and an additional domain is seen that may be attributed to M (Ge et al., 2010). (D and E) Volumes at the surface of the particle, extracted from the tomograms, revealing the presence of trimeric entities to which x-ray models of the pre-fusion (D) and post-fusion (E) structures can be manually fitted. In each case, two models (in blue and red) are displayed. (F and G) For a more quantitative fit, four trimers were isolated from the reconstructions and averaged. The x-ray models were fitted to the resulting averaged 3D reconstructions with UROX (Siebert and Navaza, unpublished program), a more user-friendly version of URO (Navaza et al., 2002). (F) For viruses incubated at pH 7.5, two fits are shown. For the fit displayed above and to the left, the crystallographic trimer, with its domains shown in color, was directly fitted in the electron microscopy reconstruction. The second fit (below and to the right) was performed with a monomer, imposing C3 symmetry. The better fit obtained with this model suggests that the trimer of G at the surface of the particle is slightly different from that in the crystalline structure. (G) At pH 5.5, the crystalline post-fusion model of G fits the tomographic model well.

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