Computational protocol: Sequence-Specific, RNA–Protein Interactions Overcome Electrostatic Barriers Preventing Assembly of Satellite Tobacco Necrosis Virus Coat Protein

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Protocol publication

[…] Diffraction data were collected at Diamond Light Source on beamline I02 at a wavelength of 0.9795 Å to a maximum resolution of 2.29 Å. High-quality low-resolution data were required to enable examination of the less ordered internal structure of the VLP. Three sweeps of data were therefore recorded from the same region of the crystal with varied detector and beamstop positions with resolution ranges of 217–7 Å, 116–3.8 Å and 30–2.29 Å. One hundred eighty degrees of data were recorded for each sweep with an oscillation angle of 1° for the very low resolution data and 0.5° for the medium- and high-resolution sweeps. All three sweeps of data were processed together using the 3dii option of the xia2 expert data reduction system (). [...] The data were isomorphous with recombinant STNV VLP (PDB entries 1VTZ and 3RQV), and this structure was used to provide initial phases for map calculation. Rigid-body refinement of the entire capsid was carried out using REFMAC5. Sixty-fold averaged maps were calculated using Coot after which the CP structure was rebuilt and then refined, using strict 60-fold NCS, with REFMAC5 (). […]

Pipeline specifications

Software tools xia2, REFMAC5, Coot
Application Protein structure analysis
Organisms Satellite tobacco mosaic virus