Computational protocol: Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus

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Protocol publication

[…] For cryo-electron tomography, the purified samples were mixed with 4X concentrated 10nm BSA gold solution (Aurion, Wageningen, Netherlands) in a 15:1 volume ratio. 3μl of this mixture was applied to Quantifoil R1.2/1.3 grids (Electron Microscopy Sciences), blotted and plunge frozen in liquid ethane.Tilt-series data was collected using a Titan Krios (operated at 300kV) and a Gatan K2 Summit detector. The Leginon software [, ] was used to collect data at appropriate grid positions. A magnification of 11000X was used with a pixel size of 1.32Å. Tilt-series were collected from -60° to +60° in 1.5° steps with a defocus range of 4–5μm for the virus samples and 5–6μm for the in vitro assembled nucleocapsid core samples. The total dose applied to each sample was between 80–90 e-/Å2 [, ].The tilt-series were aligned using gold fiducial markers and reconstructed using the IMOD software []. Ctfplotter program from the IMOD suite [] was used to estimate the defocus of each individual tilt-image in the tilt-series. CTF correction and phase-flipping of the aligned tilt-series was then carried out as part of the IMOD tomographic reconstruction procedure. Though accurate resolution estimation is challenging for tomographic reconstructions, the resolution of the virus tomograms was estimated to be better than 50 Å because it was possible to see separation between the two layers of the viral membrane in directions perpendicular to the electron beam.For negative staining, 1% solution of ammonium molybdate was added to the sample on a grid, blotted, washed twice with distilled water and then air dried after blotting off the excess liquid. The grids were analyzed using a CM200 (FEI) microscope with a 1k×1k CCD camera. [...] Sub-volumes of virions were extracted from the cryo-electron tomograms using IMOD []. The representation of the virion density was inverted using the EMAN2 suite []. Using the UCSF Chimera software [], the virion density maps were low pass filtered using a gaussian filter to 75 Å resolution, followed by removal of dis-continuous densities using the ‘hide dust’ feature. The virions were then rotated around different orthogonal axes for making a movie using Chimera []. Snapshots from these movies in color were used to make . […]

Pipeline specifications

Software tools Leginon, IMOD, EMAN, UCSF Chimera
Application cryo-EM