Computational protocol: T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

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Protocol publication

[…] All protein crystals were grown at 18°C by vapor diffusion via the “sitting drop” technique. 200 nL of each pMHC (20 mg/mL) in crystallization buffer was added to 200 nL of reservoir solution. A2/LLW crystals were grown in 0.1 M Hepes, pH7, 0.2 M ammonium sulphate, 20%PEG 4000. All crystals were soaked in 30% ethylene glycol before cryo‐cooling. All crystallization screens and optimization experiments were completed using an Art‐Robbins Gryphon dispensing robot (Alpha Biotech Ltd., UK). Data were collected at 100 K at the Diamond Light Source (Oxfordshire, UK) as described previously . All data sets were collected at a wavelength of 0.98 Å using an ADSC Q315 CCD detector. Reflection intensities were estimated with the XIA2 package, and the data were scaled, reduced, and analyzed with the SCALA and CCP4 package. Structures were solved with molecular replacement using PHASER. A solution could be obtained with a search model taken from Protein Data Bank entry 5EU5. Sequences were adjusted with COOT, and the models were refined with REFMAC5. Graphical representations were prepared with PyMOL. The reflection data and final model coordinates were deposited in the Protein Data Bank (PDB code: 5N6B). [...] The 3D structure of the TRAV12‐2/TRBV9 TCR in complex with HLA‐A2 and the LLWNGPMAV peptide was modeled from three experimental structures: 3HG1 and 4QOK , containing a complex between the TRAV12‐2/TRBC1 TCR in complex with HLA‐A2 and the ELAGIGILTV or EAAGIGILTV peptides, respectively, and the experimental structure obtained in this study for the complex between HLA‐A2 and the LLWNGPMAV peptide. The sequence alignment between TRBV9 and TRBC1 was performed using the MUSCLE program . The sequence identity between the variable part of the TRBV9 and TRBC1 TCR beta chains is 30%. Based on this sequence alignment, the model was obtained using the Modeller program , . 1000 models were generated by satisfaction of spatial restraints through minimization and simulated annealing, and the model with the best Modeller objective function was retained. Molecules were visualized and analyzed using UCSF Chimera .The model of A2/LLW in complex with the MEL5 TCR was obtained with the Modeller program, following the method described above to obtain the structural model of A2/LLW with the YF5048 TCR. In this case, however, we used our experimental structure of the A2/LLW as a template for the HLA/peptide domain and the experimental structure of the complex between A2/ELA and MEL5 (PDB code: 3HG1) as a template for the MEL5 TCR. […]

Pipeline specifications

Software tools xia2, CCP4, Coot, REFMAC5, PyMOL, MUSCLE, MODELLER, UCSF Chimera
Applications Protein structure analysis, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Yellow Fever, Substance-Related Disorders