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Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus

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Science  05 Feb 2010:
Vol. 327, Issue 5966, pp. 689-693
DOI: 10.1126/science.1181766

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  1. Fig. 1

    Cryo-EM of VSV virion and 3D reconstruction of its helical trunk. (A) A typical cryo-EM micrograph of VSV virions at 98,000× magnification. The trunk portion is marked by the boxes. (Inset) Incoherent average of Fourier transforms of all raw images showing the layer lines. (B) Density map of the virion trunk. To enhance visual clarity and to show the interior, we computationally removed four turns of M, part of the membrane bilayer, incomplete subunits, and a 30° wedge. Nucleocapsid N and matrix M layers were displayed at a threshold of 1.15 σ above the mean; envelope densities were displayed at a threshold of 0.1 σ above the mean. (C) A complete repeat of the N and M helices, featuring 75 helical asymmetric units in two turns. (D) The central, vertical slice (17.3 Å thick) in the density map. (E) A radially color-coded surface representation of a central slab (23 Å thick). 1 and 2 indicate the outer and inner leaflets of the phospholipid bilayer envelope, respectively; t indicates the putative cytoplasmic tail of G; M is the matrix protein; N is the nucleoprotein. (Inset) Maps in all panels are colored according to radial distance as depicted in the scale bar. N,red to green; M,light blue to blue; envelope membrane, purple to pink. In this and Figs. 2 to 4, the arrow in every panel denotes the directionality (tip to trunk) of virions along the axis of the helix or of parts as they would be in the virion.

  2. Fig. 2

    In situ structure of the full-length M matrix protein. (A) Fit of the crystal structure (ribbon) of the C-terminal core domain MCTD (right) of M into the corresponding density map (mesh, contoured at 1.15 σ above the mean), taken from the cryo-EM map. The α helices are shown in red and β sheets are in purple. The numbered yellow spheres in the left part of the density map mark the positions of the four contact points on the M-hub domain. The highest density regions of the cryo-EM density are shown as gray-shaded surfaces by contouring at 3.0 σ above the mean. (B) (Left) Four adjacent M (light blue) with two N (green) subunits in the neighborhood of one M with its M-hub in yellow. The contact points on the M-hub that mediate interactions with M and N are labeled 1 to 4. The volume is contoured at 1.0 σ. (Right) By turning the left panel 80° around the vertical axis and removing the frontal M subunit, the interaction between N and M is illustrated.

  3. Fig. 3

    Formation of the bullet tip of VSV virion by the nucleocapsid (N) ribbon. (A) Fitting of the crystal structure (7) of nucleoprotein (N) (yellow ribbon) and RNA (blue ribbon) into the cryo-EM density map (semitransparent green, displayed at a threshold of 1.5 σ above the mean) from the VSV virion trunk. The helical axis in this panel points toward the reader. The purple wire frames represents the highest-density regions of the cryo-EM structure (threshold of 3.5 σ above the mean), which colocalize with α helices and the vRNA of the crystal structure. (Insets) Along the upper part of the interface between adjacent C lobes in the decamer, there are six hydrogen bonds (including R309 to E419) and one (I237:Y324) hydrophobic interaction (top right inset). After flexible docking of the atomic structure from the decamer into the cryo-EM density map of the trunk, distances between amino acid partners in these seven sites increase by ~9 Å, disrupting these interactions. (B) Comparison of the inclination of N subunits (green) in our cryo-EM structure from the trunk of the virion (37.5 subunits/turn) with the inclination of the N subunits (red) in the crystal structure from the decamer ring (10 subunits/turn) (7). (Top) Dashed lines through a side view of an N subunit from the trunk (left) and with an N subunit from the decamer (right) show the difference in tilt, the angle up from the horizontal plane. (Bottom) Dashed lines through end-on views of N subunits show the difference in dihedral angle between adjacent N subunits in the trunk (green) and in the decamer ring (red). (C) A representative class-average of the virion tip from 75 individual images. Numbers inside the nucleocapsid designate the order of N subunits in the nucleocapsid ribbon, which may be traced by following the path 1 > 1a > 2 > 2a, etc. (D) Negative-stain EM images of the wild-type decamer and two mutant rings confirm the importance of two of the interactions specified above. Both mutants produce rings larger than a decamer. (E) An illustration of a plausible process by which the nucleocapsid ribbon generates the virion head, starting with its bullet tip. The curling of the nucleocapsid ribbon generates a decamer-like turn at the beginning, similar to the crystal structure. When assembly nearly completes this turn, continuation of vRNA requires that the ribbon form a larger turn below it, similar to that in the mutants in (D). As the spiral enlarges and progresses to the helical trunk, the tilt of individual N subunits decreases. When it reaches the seventh turn, the nucleocapsid ribbon becomes helical (insets), in which each new turn of the nucleocapsid fits naturally under the preceding turn (insets).

  4. Fig. 4

    Architecture of the VSV virion. (A) Representative 2D averages of conical tip, trunk, and base of VSV and a montage model of the tip and the cryo-EM map of the trunk. N is green, M is blue, and the inner (2) and outer (1) leaflets of the membrane are purple and pink, respectively. (Inset) Illustration of the base region of the VSV virion. The “X” marks the absence of a turn of M helix below the lowest turn of the N helix. (B) Cryo-EM structure showing the putative cytoplasmic tail of G protein binding to an M subunit through a thin linker. The inner leaflet (2) of the membrane has unusual bumps (arrowhead) that meet M at the site of a thin linker density (arrow). (C) A wedge of the virion trunk, illustrating its geometric arrangement across the three layers. The N layer, the M layer, and the two membrane leaflets (1 and 2) are arranged in coaxial cylinders with their radii determined from our cryo-EM structure. Because of the difference in radii, the helical lattice points on the three layers form different triangles. The smaller the radius, the narrower the apex angle (inset). At the outer surface of the membrane, the lattice points form an equilateral triangle.