Computational protocol: The infectious particle of insect-borne totivirus-like Omono River virus has raised ridges and lacks fibre complexes

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Protocol publication

[…] Purified and further concentrated OmRV (1.43 mg/mL) was embedded in vitreous ice and examined at liquid nitrogen temperature with a cryo-EM (JEM2200FS; JEOL) operated at an accelerating voltage of 200 kV and at a nominal magnification of × 50,000. An omega-type energy filter was used to obtain a zero loss electron beam (20 eV slit width). Images were recorded on a 4k × 4k charge-coupled device (CCD; TVIPS) with an electron dose of ∼20 e−/Å2 with applied underfocus values of approximately 1–3 μm and at pixel size of 1.88 Å/pixel. EMAN2 was used for selecting individual viral particles. Empty and dsRNA-full particles were boxed separately. Contrast transfer function (CTF) and amplitude corrections were automatically performed on boxed particles by e2ctf.py (EMAN2) and then manually verified by adjusting defocus and B-factor values. Several random icosahedral initial models were calculated by 15 class-averaged OmRV particle images, each calculated using 300 raw particle images. A total of 12,207 empty and 6,224 full particles were aligned and classified to reconstruct the two 3D structures using EMAN2 and RELION. One calculated random model was selected and used for reference-based iterative refinement using icosahedral symmetry by EMAN2 to obtain an initial 3D reconstruction. After 10 iterative steps of 3D refinement, the first refined model was computed from empty OmRV particles and used for 3D refinement using RELION. In RELION, CTF and amplitude corrections of images were automatically performed by ctffind3. Good particles were extracted from good 2D class-averaged images and used for maximum likelihood-based 3D reconstruction using RELION. The final resolution of the empty OmRV model was estimated to be 8.3 Å (FSC cutoff 0.143). A full particle was reconstructed from a particle set of dsRNA-full particles by using the final empty OmRV structure as an initial model using RELION. Segmentation analyses of OmRV and IMNV capsid subunits were conducted manually with the assumption that the two chemically identical capsid protein subunit types should roughly share the same shape. The two subunits were separately extracted from the overall structure by masking away all density clearly belonging to neighbouring subunits. The structures of the two subunits were then superimposed and the final subunit structure was determined by using the identity between the two subunits using Chimera as previously described. The 3D volumes and cross sections of OmRV, IMNV and other totiviridae viruses were visualized by Chimera and Segger software. The 3D maps were rendered at an isodensity contour level of 2.6 σ and 2.1 σ (equivalent to numerical map values of 0.0900 and 0.0938) in empty and full particles. in Chimera. The radial sections were generated by bsoft (bradsec). [...] The cleavage sites of the OmRV and IMNV CPs were predicted in previous articles; MS/MS identification of the OmRV major CP in this study agreed with those predictions (see ). The predicted amino acid sequences of the major CPs were aligned by the T-coffee multiple sequence alignment server. Additionally, the sequences were applied to the PSIPRED protein sequence analysis workbench for predicting secondary structures. […]

Pipeline specifications

Software tools EMAN, RELION, CTFFIND, Segger, Bsoft
Application cryo-EM
Organisms Human poliovirus 1 Mahoney, Viruses