Computational protocol: A higher-order entity formed by the flexible assembly of RAP1 with TRF2

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Protocol publication

[…] Crystals of TRFH in complex with peptide RAP1-YLP 89ENRERLELEAYRLGPASA106 were obtained by hanging drop vapour diffusion at room temperature, with a concentration of TRFH of 34 mg/ml and a molar ratio of TRFH/Rap1-YLP of 1:3.5 in a buffer containing 12.5 mM Tris pH 8.0, 100mM NaCl and DTT 2.5 mM. The most suitable crystals grew in 16% PEGmme 550, 50 mM Hepes pH 8.0, 5 mM MgCl2, 5 mM DTT. Native diffraction data were collected on PX1 beamline at SOLEIL synchrotron, and reduced with XDS (). The structure was solved by molecular replacement with PHASER (), using one TRFH monomer from 3BUA PDB entry: as model probe (), and refined with BUSTER5 (). [...] The BRCT domain of RAP1 was modelled using MODELLER software () and the structure of the BRCT domain of Rap1 from S. cerevisiae (, PDB entry 2L42). The resulting model and Myb domain of RAP1 () were then manually positioned along the TRFH domain of TRF2 with Pymol (, based on the TRFH/RAP1-peptide crystal structure and the protein footprinting results. Conservation analysis was performed with CONSURF software () using 14 sequences for both TRF2 and RAP1 from the same species of vertebrates (See sequence codes in Supplementary Data). […]

Pipeline specifications

Software tools XDS, MODELLER, PyMOL, ConSurf
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Liver Diseases, Neoplasms