1. Volume 5, Issue 2, Pages 255-266 (February 2000)
Cryo-Electron Microscopy Reveals the Functional Organization of an Enveloped Virus, Semliki Forest Virus 
Erika J Mancini, Mairi Clarke, Brent E Gowen, Twan Rutten, Stephen D Fuller 
Molecular Cell 
Volume 5, Issue 2, Pages (February 2000)
DOI: /S (00)

2. Figure 1 Overview of the SFV Structure
(A and B) A comparison of the 9 Å (A) and the 22 Å (B) (Fuller et al. 1995) reconstruction. Depth-cued surface representations of the reconstructions are viewed along the icosahedral two-fold axis. The color scheme reflects the radial distance from center, increasing from blue to red, as shown in the inserted color scale. Scale bar, 100 Å.
(C and D) Regions of cEMs of SFV virions used for image processing and three-dimensional reconstruction. The effect of defocusing on the appearance of viral features is shown for δf = −1 μm (C) and δf = −3 μm (D). Scale bar, 500 Å.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2
Correspondence between Order and the Features of the Reconstruction
(A) A central section of the virus surface view reveals the internal organization of virus. The RNA (R) (shown in yellow) is enclosed in an icosahedral nucleocapsid made of 240 copies of protein C (C) (shown in red). The nucleocapsid is enveloped by a lipid bilayer (M) (inner and outer leaflets shown in blue). The lipid bilayer is covered and penetrated by 80 glycoprotein spikes (S) (shown in white).
(B) Radial dependence of the Fourier shell correlation. The Fourier shell correlation (FSC) (Harauz and van Heel 1986) was calculated between 35 Å thick concentric shells of two independent reconstructions and plotted as a function of the radius. The spacing for which the FSC reaches 0.5 (dashed red line) varies with the radii, between 10.4 Å and 22 Å. The correspondence between this variation and the internal features of the virus is evident in the plot of the radial normalized density average (solid green line).
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 Features of the Nucleocapsid
(A) A surface view of the nucleocapsid density along the icosahedral two-fold axis is shown for the density between r = 169 Å and r = 198 Å. The 240 copies of the C protein monomer are arranged in a T=4 lattice with pentamer–hexamer clustering. Notice the departure from quasiequivalence of the hexameric subunits as shown by the bend of the hexamer (line). The four independent subunits of the asymmetric unit are marked with a, b, c, and d. Scale bar, 100 Å.
(B) The fit of the atomic structure of the C protein (Choi et al. 1996) to the observed capsid density. A real space fit was performed for each of the four independent positions of the asymmetric unit using the program emfit (Cheng et al. 1995). The C protein crystal structure is represented as a ribbon diagram. The position of the N terminus for the four independent subunits is marked with a yellow asterisk. Scale bar, 25 Å.
(C) Surface view of the density corresponding to the d subunit, showing details of the fit of the C protein monomer (Choi et al. 1997) into the observed density. The density shown corresponds to that between the RNA (R) (r = 145 Å) and the inner leaflet (IL) of the lipid membrane (r = 225 Å). The C protein crystal structure is represented as a ribbon diagram with the same color code as in Figure 3B. Scale bar, 25 Å.
(D) Surface view of the GAP average of the four independent C protein monomers showing the internal cavities and the position of the arm. The residues of the hydrophobic pocket (Choi et al. 1997), which is the putative binding site for the E2 protein carboxy-terminal tail, are represented in a green. Residues Tyr-184 and Trp-251, which are situated at the entrance of the hydrophobic pocket, are colored in pink. Notice how the low-density region in the average density corresponds to the cavity created by the hydrophobic pocket. The 16 residues, which connect the first residue visible in the crystal structure (Cys-119) with the body of the protein, are represented as a red ribbon. This loop in the structure of the isolated C protein (Choi et al. 1997) lies adjacent to but outside the arm of density connecting the subunits. Scale bar, 12 Å.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4 Transmembrane and Skirt Regions
(A) Transmembrane regions and interaction with the capsid. (Left) Surface representation of the density around the paired transmembrane segments that is above the c capsid subunit. The C protein is shown as a ribbon diagram with the coloring as in Figure 3C. The positions of the inner leaflet (IL, radius = 213 Å) and the outer leaflet (OL, r = 261 Å) of the membrane are marked. The putative α-helical transmembrane segments for E1 and E2 are depicted with the paired helical segments of Rop (residues 1–25 and 31–56 of 1ROP) (Banner et al. 1987), demonstrating that the dimensions and the topology of the density are consistent with a pair of helices. (Right) The sections show the density at the positions indicated by the arrows. The transmembrane domains are seen as paired rods of density approximately 10 Å wide separated at the top by approximately 10 Å (top panel) and twist about each other in a right-handed sense. Scale bar, 15 Å.
(B) Skirt and subskirt region. (Left) Surface representation of the density corresponding to the skirt and subskirt regions near a quasi-six-fold (icosahedral two-fold, 2f) axis. The positions of the outer leaflet of the membrane (OL, radius = 261 Å) and the skirt (SK) are marked. (Right) The sections show the density at the positions indicated by the arrows. The subskirt region is spanned by twisted rods of density approximately 20 Å long and approximately 10 Å wide. The skirt itself is a 15 Å thick, well-ordered layer of density with features that may represent protein domains. Scale bar, 10 Å in the left panel and 45 Å in the right panel.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5 The Structure of the Projecting Domain of the Spike
(A and B) A radially depth-cued surface view of a three-fold spike and its connection to the underlying skirt region seen from the outside (A) and the complementary view from the membrane (B).
(C) A side view of the surface of the three-fold spike and underlying skirt region with the positions of the sections indicated by the arrows. Notice the separation between the E1-E2_E3 heterotrimers within a spike, the separation between two main peaks of density within the heterotrimer, and the presence of a cavity in the middle of the spike. The section through the skirt region reveals the switch between the trimer clustering of the spikes to the pentamer–hexamer clustering of the capsid.
(D) Central section through a three-fold spike. The density is represented in blue (lower contour level) and red (higher contour level). A 10 Å wide high-density region is seen at the edge of the central cavity. The center of the cavity is marked by a yellow asterisk in (B) and (D) and by an arrow in (C). Scale bar, 10 Å.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6
Overview of the Features of the Spike and Capsid in the Virion
This composite shows the relative placement of the features in the 9 Å reconstruction of SFV. The RNA is the least well-ordered region of the structure, although a shell of density just below the C protein appears to be localized by interactions with it. The capsid protein is well ordered and penetrates the inner leaflet of the bilayer where it interacts with the carboxy-terminal tails of E1 and E2. The E2 tail corresponds to a density in the map that lies above a hydrophobic pocket in the capsid protein. The transmembrane regions are visible as pair of rods of density twisting in a right-handed sense as they cross the membrane. This is a relatively poorly ordered region of the map. A twisted pair of rods of density connects the transmembrane regions to the well-ordered skirt. The hollow projecting region sits upon the skirt and forms a dome 60 Å above the membrane. Low-density regions mark the separation between the three E1-E3_E3 heterotrimers in the spike and match the positions of seams in the skirt. Three striking high-density features border the 40 Å wide cavity and extend from the skirt to the top of the spike. We have represented one of these in red to indicate its position in the spike and path through the structure without trying to suggest its secondary structure.
Molecular Cell 2000 5, DOI: ( /S (00) )