Computational protocol: 4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

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Protocol publication

[…] We carefully screened all the CCD frames from which 3558 images with evident signals up to 1/5 Å−1 in their 1D power spectra were selected for subsequent processing. A total of 37 315 virus particles were automatically boxed out using ethan (), among which ∼10 000 particles contained continuous carbon film. The contrast transfer function parameters for each CCD image were manually determined using ctfit in EMAN1 (). An initial model at ∼7 Å resolution was quickly obtained by MPSA (). The structure was further refined by EMAN1 using standard projection matching method with progressively decreasing angular step size (with a final value of 0.4°). After each iteration, the non-icosahedral part, including the lipids and the RNA, in the reconstruction was removed by a soft-edged mask, which defines the outline of the icosahedrally organized, low-pass filtered ‘protein-only' content in the map. This masked map then served as the reference model for the next iteration. The resolution of the final reconstruction was estimated to be 4.8 Å based on the 0.5 criterion of the Fourier shell correlation (FSC) between two independent reconstructions (; ). [...] To model the VEEV E1, E2 ectodomains and E3 (E1: residues 1–389; E2: residues 1–341 and E3: residues 1–59), the sequence alignment and subsequent homology modelling was performed by MODELLER (), using the crystal structure of its CHIKV homologue () (PDB ID: 3N40) as the template. The missing parts in the crystal structure (E1: residues 390–442; E2: residues 342–423) were modelled de novo by first tracing the backbones using GORGON (), with several visible side-chain densities serving as the anchor points.To model the TM helices, in particular, we generated the Cα models of two consecutive helices (residues 403–412 and 415–442) separated by a kink for E1, and a long straight helix (residues 367–401) for E2. These residue assignments are based on both the secondary structure predictions from PSIPRED online server (; ) and our cryo-EM density map. The three helical models were placed in the corresponding densities in GORGON, and the registration of their Cα positions was determined by the evident bulky side-chain densities along the helices (e.g., E1-W407, E1-W409, E1-Y434 and E2-W387).Next, we converted all the de novo traced Cα models to their corresponding all-atom models of VEEV using SABBAC online server (), and then stitched the homology and de novo portions together in COOT () to generate the initial full-length E1 and E2 atomic models.Our model for the CP protease domain is taken directly from the previous crystal structure of VEEV TC-83 (PDB ID: 1EP5: A, residues 120–275), and was fitted into the density map as a rigid body using CHIMERA's Fit to Map function. The α-helix of CP (residues 115–124) was modelled de novo in the same way as the E1 and E2 TM helices.Finally, we used ROSETTA () to refine the full-length E1, E2 and the structured part of CP. The E3 homology model was not further refined due to the less-resolved quality of our E3 density. ROSETTA uses the cryo-EM density as a restraint, along with energy minimization to eliminate the steric clashes and assure proper molecular geometry. In total, two separate refinements were performed. First, one set of E1-E2-CP molecules was refined against the asymmetric unit averaged map. Second, four copies of the models from the first round were placed at the T=4 related positions within the asymmetric unit, and these four sets of E1-E2-CP molecules were refined together against the original cryo-EM density map to produce our final model. […]

Pipeline specifications

Software tools EMAN, MODELLER, Gorgon, PSIPRED, SABBAC, Coot
Applications cryo-EM, Protein structure analysis
Organisms Equus caballus, Venezuelan equine encephalitis virus, Homo sapiens
Diseases Encephalomyelitis, Venezuelan Equine