The 3D cryo-EM reconstructions, cryo-ET reconstructions, 3D models of the Ebola virus, and the atomic resolution structure 3CSY.pdb were visualized using UCSF Chimera software package (Computer Graphics Laboratory, University of California, San Francisco, supported by NIH P41 RR-01081) [53] . The 3D images and movies presented in this manuscript were generated directly by UCSF Chimera software package. , Region three is shown, with the locations of correlation maxima shown with an X. The angular distance between each maximum was calculated from several plots. A total of 71 measurements gave an average angular distance of 33.6u+/ 28.5u between helical repeats, resulting in 10.7 repeats per turn. Using the 6.96 nm pitch (Fig. S8 ) the step in Z per helical repeat was calculated as 0.65 nm. These helical symmetry values were then imposed on the nucleocapsid tomogram and this structure was used as the initial reference volume for refinement using the iterative helical real space reconstruction method. (TIF) Figure S3 Surface spike distribution in the Ebola VLP. Longitudinal Z-slices through the top and middle of the particle are shown, as well as the end-on view (A). The tomogram is shown as a shaded surface at a density threshold that indicates the spikes (B). The volume from one side of the tomogram has been extracted, and a red-blue color scheme shows the depth at which the spikes are located. This region of the envelope has a surface area of 15,651 nm 2 . Selected spikes have been identified by red circles, the single particle reconstruction of the spike is shown at the same scale to the right in a red square for comparison. The same region in (B) is shown in (C) with a solid orange cylinder to provide a visual cue for the viral envelope. Eighty-six individual spikes were counted (white spheres) and have a patchy distribution (D), each spike would occupy an average area of 182 nm 2 , giving an average spacing between spikes of 15.2 nm. The reconstruction of the spike (blue) with the docked KZ52 Fab (purple) has been included to show that there is ample room for antibody attachment. (TIF) Figure S4 Extraction of Ebola nucleocapsid structure for sub-tomographic analysis. The tomogram of a linear region of the Ebola virus was used as the first reference for subtomogram analysis (A). When viewed along the helical axis (Y) or from the end perspectives (X,Z) the basic components are visible. The tomographic volume was also cylindrically masked along the X-axis, selecting only the density containing the nucleocapsid, to highlight the components of the nucleocapsid in the tomogram (B). Two-dimensional single particle image analysis was carried out with cryo-images (C) (not tomographic data sets), for comparison to the 3D tomographic data. The average shown in this panel was generated by reference free classification, using the ''startnrclasses'' program in EMAN [54] . The 6.96 nm helical pitch can be easily seen in the 2D average, but is also visible in the projections of the tomographic volume in (A, B) . (TIF) Figure S5 Representative low-magnification images of Ebola virus. Frozen hydrated virus is clearly visible with sections of the filamentous virus over both the support film and across the holes in the quantifoil film. Individual G1 (single genome copy) virus is circled in red, several sections containing a nucleocapsid are indicated by a blue arrowhead, and regions without a nucleocapsid are indicated by a magenta arrowhead. Globular heads are identified by yellow arrowheads. In this image the circles (light grey, 2 m diameter) are filled with frozen hydrated virus in a thin aqueous layer, and the quantifoil support film appears as darker grey.