Computational protocol: Mutations in VP1 and 5′-UTR affect enterovirus 71 virulence

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Protocol publication

[…] Purified EV-V and EV-R were inactivated by UV light exposure and prepared using a standard cryoEM procedure. In short, one drop (~3 µL) of inactive virus in 2% glutaraldehyde buffer was applied to glow-discharged TEM copper carbon-coated grids (Electron Microscopy Sciences) and mounted onto the plunge freezing device (Gatan CP3). The sample with the grid was frozen in the liquid ethane and then transferred to liquid nitrogen for further image collection. TEM imaging was performed with a JEOL1400 TEM at a magnification of 30,000× and an acceleration voltage of 120 kV. All digital images were acquired with a Gatan, Inc. Ultrascan 4000 4 k × 4 k CCD Camera System (Model 895). Virions harvested from RD and Vero cells were selected manually using the package in EMAN2. ML2D, an image classification algorithm package included in Xmipp 3.1, was used to classify the selected particles as empty or full. The crystal structure of the EV-VP1 protein (PDB ID code: 3VBS) was manually fitted into the mature EV71 cryoEM structure (EMDB ID code: 5558) using UCSF Chimera. […]

Pipeline specifications

Software tools, EMAN, Xmipp, UCSF Chimera
Application cryo-EM
Organisms Homo sapiens, Mus musculus
Diseases Hand, Foot and Mouth Disease
Chemicals Nucleotides