We identified filamentous EBOV particles 20 microns or longer, with a well ordered internal structure, and a helical nucleocapsid giving an internal ''herring-bone'' appearance using cryo-EM and cryo-ET (Figures 1, 2 , S1, S5). The nucleocapsid, as observed within intact viral particles, has a uniform helical structure (Figures 1, 2, 3, 4) and is enveloped by a membrane coated by an external layer of GP spikes. From the same image data set, we combined extracted volumes from tomograms with 2-D single particle processing to determine the structure of the GP spikes ( Figure 5 ) to a resolution of 14 Å as measured by the Fourier Shell Correlation (FSC) 0.5 criterion. Virions are rarely straight. Variation in the overall length of virions is non-random, and they fit into ordered size classes ( Figure 1 ). Analysis of 2090 distinct intact virions with a nucleocapsid from cryo-electron micrographs shows that the most common class length (53%) of virus particles is 982679 nm ( Figure 1A , Table S1 ). The other size classes are multiples of this length. Enveloped filovirus particles have several Empty and linked EBOV structures were excluded from the histogram data. A single G1-single/comma shaped EBOV is shown (inset on the right, G1 = 1 copy of genome). (B) Low magnification cryo-images showing: G1-single/comma shape, G1-single/ linear, G5-continuous (G5 = 5 copies of genome). (C) High magnification of a G1-(single genome) virion with a region filtered to emphasize the nucleocapsid. (D) Low magnification image of a G4-linked EBOV, each genome copy is indicated and numbered, the red arrows show the transition points between nucleocapsids. The circular holes (filled with vitreous ice) appear as lighter regions and the support film (''quantifoil'') appears dark grey. A ''linker'' region is shown at higher magnification (inset). doi:10.1371/journal.pone.0029608.g001 different morphologies (shown in Figures 6, S1 , S5). These configurations are ''single'' particles, containing a nucleocapsid of uniform length, (which we postulate to contain one copy of the genome), ''continuous'' particles, with nucleocapsids of a length of the single virion multiplied by an integer of 2 or greater; and ''linked'' virions composed of a series single-genome nucleocapsids connected by short sections of empty envelope. Negative staining can cause drying and staining artefacts which hamper accurate measurements and preclude 3D analysis. Nevertheless, in previous studies using this technique, the average length of Marburg virus Figure 2 . Image processing of Ebola virus. Linear 2D averaging of EBOV: the envelope and nucleocapsid are prominent features (A). The line trace is colour-coded as follows: red, spike; beige, lipid envelope; green, membrane-associated proteins; white, membrane-nucleocapsid gap; blue and purple, outer and inner nucleocapsid. (B) 2D class averages of envelope plus inner face. (C) VP40 VLPs, showing 2D averages from the from side regions (first two) and end-on/central regions (last three). In (A-C) representative individual repeats have been highlighted in color using the same scheme as in (A). (D) Schematic model of the nucleocapsid and envelope, highlighting the relative distribution of NP to VP40. (E,F) 3D reconstruction of the nucleocapsid with the same colour scheme as in (A). The location of the inner nucleocapsid, and the bridge are indicated. The reconstruction is presented at a volume threshold that would encompass a single copy of each of these proteins, and the viral RNA. In (E) the vertical (protein-protein) and horizontal (protein-RNA) contacts are indicated by yellow and white arrows, respectively. (G) Various recombinant nucleocapsid-like structures, and authentic EBOV, which have been studied by electron microscopy [14, 15, 21, 28] . 3D schematics of these structures highlighting the RNA and protein composition and the diameter of these structures, at the same scale for comparison to (E). doi:10.1371/journal.pone.0029608.g002 was measured as 790 nm, and EBOV as 970 nm [21, 22] : the latter is very similar to the mean length of 982 nm measured in the current study by cryo-TEM of frozen hydrated specimens. In addition, discrete sub-populations of virions of double or triple the average length were also previously reported in centrifuged virus preparations [21] .