Computational protocol: Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers

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Protocol publication

[…] First crystals were found in 0.1 M Tris-AcOH, pH 8–8.5, 0.2–0.4% (w/v) Cystamine, 0.2 M LiSO4, 8% (w/v) PEG 20’000, 8% (w/v) PEG 550 MME using sitting drop vapor diffusion at 20 °C. TRIM20Δ413 (7.5 mg/ml) was mixed in a 1:1 ratio with mother liquor. The obtained crystals were used for micro-seeding in 0.1 M Tris-AcOH, pH 7.4–8.0, 0.26% (w/v) Cystamine, 0.2 M LiSO4, 8% (w/v) PEG 20’000, 8% (w/v) PEG 550 MME using sitting drop vapor diffusion at 20 °C. The protein sample was concentrated to 5 mg/ml and mixed in a 1:1 ratio. Crystals grew within 24 h and were harvested within 18 days. Prior to flash freezing in liquid nitrogen, crystals were soaked in the mother liquor supplemented with 20% (v/v) ethylene glycol. A native data set of a TRIM20Δ413 crystal was recorded at the X06SA beam line of the Swiss Light Source (Paul-Scherrer Institut, Villigen, Switzerland). The protein crystallized in P21 with 6 molecules in the asymmetric unit forming 3 dimers. Data was indexed, integrated and scaled with XDS to a resolution of 2.4 Å. Molecular replacement was done with Phaser using the B30.2 domain of TRIM20 as search model (pdb entry: 2wl1). Model building and manual fitting was done in Coot. Refinement was performed with Phenix. In the final model, 98% of all residues are in the favored Ramachandran area and no outliers were detected. […]

Pipeline specifications

Software tools XDS, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Human immunodeficiency virus 1, Dipturus trachyderma
Diseases Familial Mediterranean Fever, Hereditary Central Nervous System Demyelinating Diseases, Genetic Diseases, Inborn