Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions

Abstract

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

Fig 1. Typical TEM micrograph showing ϕ6 NCs to be selected for boxing.

Fig 2. Illustration of the use of a component tree for structural analysis using part of NC component tree to show relationship between nodes of the tree and image voxels.

Fig 3. Component tree of a 1D example.

The nodes are indicated by black disks. The horizontal bars through the disks identify voxels that belong to the sets of voxels associated with the corresponding nodes and the numbers next to the disks are the numbers of voxels in those sets.

Fig 4. Near central slice of the NC reconstruction showing the NC elements.

Fig 5. NC Fourier Shell Correlation.

Resolution is 1.1 nm using an FSC cutoff of 0.143.

Fig 6. Class Analysis of dsRNA projections without imposed symmetry.

The 20 classes have a comparable number of particles in each class indicating a non-symmetric reconstruction could not properly present the RNA. Bar is 10 nm.

Fig 7. Simplified component tree of the NC reconstruction with branches representing the viral elements identified and labeled in the tree.

Fig 8

Isosurfaces of viral elements generated from selected voxels in the NC component tree branches identified as (A) P8 and P4; (B) P1; (C) L and (D) M segments of dsRNA and external P2; (E) S segment dsRNA; (F) internal P2 and P7.

Fig 9

A) Series of NC reconstruction slices with offset distance from central slice indicated at left. Voxels corresponding to component tree branches are highlighted for: B) L-RNA segment (blue); C) M-RNA segment (red); D) S-RNA segment (cyan); E) P2 (green); and F) P7 (yellow).