Computational protocol: Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding

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Protocol publication

[…] For crystallization, MAGE-A3 and MAGE-A4 were concentrated to 5 mg/ml and 9 mg/ml respectively and sitting drop vapour diffusion crystallization trials were set up with a Mosquito (TTP Labtech) crystallisation robot. Crystals of native MAGE-A4 were observed to grow at 4°C from conditions containing 2.6 M NaCl, 0.1 M Tris-HCl pH 8 and were cryoprotected by transfer to a solution containing 20% (w/v) D-glucose prior to being loop mounted and plunged into a pool of liquid nitrogen. Diffraction data was collected at Diamond light source beamline I04, and processed using MOSFLM []. SeMet-substituted MAGE-A4 crystallized in 0.3 M Na-malonate pH 7, 20% PEG 3350, 10% ethylene glycol and 0.1M Bis-Tris propane pH 8.5, and crystals were cryo protected in a reservoir solution supplemented with 25% ethylene glycol. A two wavelength MAD data set was collected at the Swiss Light Source (SLS) on beamline X10SA at λ = 0.9782 Å (peak) and 0.97105 Å (remote). Data were integrated in MOSFLM[], an SeMet positions located using SHELXD[] and refined using program autoSHARP[]. The initial model was built using BUCCANEER[] and extended and completed manually using COOT[]. The final refinement was performed using the 2.3 Å native dataset and the REFMAC[] program to a final Rfactor = 24.3%, Rfree = 27.4%.MAGE-A3 crystallized at 4°C in conditions containing 15% PEG 3350, 0.1M Mg Formate, and crystals were loop mounted and cryo protected in a reservoir solution supplemented with 25% ethylene glycol. Data were collected to 2.0Å resolution at Diamond light source beamline I24, and processed using XDS[]. The structure was solved by molecular replacement using the program PHASER[] and the structure of MAGE-A4 as a search model. Refinement was performed using PHENIX REFINE[] to a final Rfactor = 22.3%, Rfree = 23.4%. A full summary of the data collection and refinement statistics are found in . [...] Small angle X-ray scattering measurements of MAGE-A3 in solution were performed at Diamond light source beamline B21 using a BIOSAXS robot for sample loading. Measurements were made using three different protein concentrations of 3.5, 1.75 and 0.88 mg/ml respectively in a buffer comprising 10 mM Hepes pH 7.5, 250 mM NaCl, 0.5 mM TCEP. The data were reduced and buffer contributions subtracted with the DawnDiamond software suite and analysed using the program SCATTER ( Pairwise distance distribution P(r) functions were calculated using GNOM[] and compared to theoretical P(r) distributions from model coordinates in calculated in SCATTER. Real space scattering profiles of atomic models were calculated from atomic models using CRYSOL[] and aligned and scaled to the experimental data using PRIMUS[]. […]

Pipeline specifications

Software tools iMosflm, SHELX, Buccaneer, Coot, XDS, PHENIX, ATSAS, CRYSOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Melanoma, Neoplasms