Computational protocol: Plate Tectonics of Virus Shell Assembly and Reorganization in Phage Φ8, a Distant Relative of Mammalian Reoviruses

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Protocol publication

[…] Crystallization trials were performed by hand in 24-well Linbro plates using the sitting-drop vapor-diffusion method at 15°C, mixing 1 μl of protein solution with 1 μl reservoir solution and equilibrating the drop against 1,000 μl reservoir solution. Native and selenomethionine-labeled Φ8 P1 crystals appeared within 1 week of setup in 100 mM Tris [pH 8.5], 30 mM sodium citrate, and between 34% and 39% PEG 200. Mercury incorporation was obtained by soaking the crystals in a drop containing Baker’s dimercurial (1, 4-diacetoxymercuri-2, 3 -dimethoxybutane) at a final concentration of 3 mM for 60 min; crystals were then back-soaked against the reservoir solution containing 20% glycerol. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Diffraction data were indexed, integrated, and scaled with HKL2000 (). The crystals belonged to space group P41212 with unit cell parameters a = b = 313.1, c = 524.8Å, α = β = γ = 90° ().The P1 structure was solved using a single anomalous dispersion (SAD) experiment at the peak wavelength of the mercury X-ray absorption edge. HKL2MAP () was used to detect heavy atom sites, which were then input in PHENIX AUTOSOL (), resulting, after density modification, in a low-resolution (6 Å) interpretable electron density map. Inspection of the map revealed two pentamers of P1 in the crystallographic asymmetric unit, clearly visible due to the unusually high solvent content (80%). The portion of the map corresponding to the asymmetric unit was cut out, placed in an oversized P1 cell, and used for molecular replacement using PHASER () with both the selenomethionine-labeled Φ8 P1 data set (at 4.8 Å resolution), in order to find the selenium site positions, and the higher resolution (3.7 Å) native data set, for phase extension. Initial manual building with polyalanine chains was carried out with the program COOT (), and the sequence assignment was facilitated by the location of selenium sites. Because of the excellent observation-to-parameter ratio of 4.5, arising from the 10-fold NCS and extremely high solvent content, it was possible to use phase and NCS-restrained individual atom refinement with REFMAC5 (), which resulted in final Rwork and Rfree of, respectively, 24.5% and 25.8% and good stereochemistry. The final structure was validated with MOLPROBITY (), and detailed data collection and refinement statistics are presented in . […]

Pipeline specifications

Software tools HKL2MAP, PHENIX, Coot, REFMAC5, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens