Computational protocol: The Smallest Capsid Protein Mediates Binding of the Essential Tegument Protein pp150 to Stabilize DNA-Containing Capsids in Human Cytomegalovirus

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Protocol publication

[…] An aliquot of 2.5 µl purified sample was applied to a 300 mesh Quantifoil R1.2/1.3 grid, blotted with filter paper, and plunge-frozen in liquid ethane. CryoEM images were collected at liquid nitrogen temperature in an FEI Titan Krios cryo electron microscope operated at 300 kV with parallel illumination. The wild type HCMV virion and SCP-deficient HCMV particle images were recorded on a Gatan 4k×4k charge-coupled device (CCD) camera at an effective magnification of 97, 498× (nominal magnification 59, 000× on film plane), corresponding to an effective pixel size of 1.538 Å/pixel at the specimen level. The HCMV capsid images were recorded on Kodak SO-163 films at a magnification of 59,000× and micrographs were digitized with Nikon Coolscan 9000ED scanner at a step size of 6.35 µm/pixel, giving a pixel size of 1.076 Å/pixel on specimen. In all cases, the electron dosage used in cryoEM imaging was ∼25e−/Å2. The defocus values were determined with CTFFIND and are in the range of 0.5 µm to 3 µm underfocus.Data processing and 3D reconstructions were accomplished with IMIRS , . Orientation and center parameters of each particle were refined against projections computed from 3D reconstructions in an iterative procedure until no further improvement in the reconstruction was obtained. Particles were selected based on the phase residues between the images and the projections. 3D reconstruction was obtained using the symmetry-adapted spherical harmonics method . The final capsid and virion reconstructions were obtained by averaging 20,502 particles (selected from 37,460 capsid images) and 11,863 particles (selected from 56,297 virion images), respectively.Visualization and averaging of density maps were carried out with UCSF Chimera . Density regions to be averaged were segmented out as density cubes of similar size. These density cubes were then first manually aligned and subsequently computationally aligned by the “fit in map” function of Chimera. Averaged density was produced by executing the “vop add” command on the above aligned density cubes.Secondary structure prediction of pp150 was performed with PSIPRED using the Protein Structure Prediction Server . […]

Pipeline specifications

Software tools CTFFIND, IMIRS, UCSF Chimera, PSIPRED
Application cryo-EM
Organisms Human betaherpesvirus 5, Homo sapiens, Human alphaherpesvirus 1
Diseases Abnormalities, Drug-Induced, Cytomegalovirus Infections, Protein Deficiency