Computational protocol: Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases

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Protocol publication

[…] X-ray diffraction data were collected at beam line BL44XU of SPring-8 (Harima, Japan), and processed using the program HKL2000[] to the resolutions of 1.90 Å and 2.30 Å for native and SeMet crystals, respectively. Molecular replacement and the phase improvement with solvent flattening were performed using the program MR-Rosetta[]. The initial electron density map of the native data set obtained by the molecular replacement using the structure of the maltose-free form of MBP [PDB: 1PEB] as the search model showed discontinuous electron densities in the region corresponding to the C-terminal domain of AfAglB-L. Then, we calculated the electron density map using phases obtained by molecular replacement combined with SAD phasing, but the quality of the electron density map did not improve significantly. Further manual model rebuilding was performed with the program COOT[], and subsequent crystallographic refinement was performed with the program PHENIX[]. Fortunately, the positions of nine selenium atoms in selenomethiones in the C-terminal domain of AfAglB-L were clearly visible in the anomalous difference Fourier map calculated from the Se-SAD data set. The superposition of the coordinates of AfAglB-S1 [PDB: 3VGP] and AfAglB-S2 [PDB: 3VU0] onto the partially built model of the N-terminal α-helix of the C-terminal globular domain of AfAglB-L correctly placed the AfAglB-S1 and -S2 structures in the electron density maps. Because the CC unit is common in all AglB and PglB proteins, the superposed structures guided the manual model building and refinements to obtain the final model of the C-terminal domain of AfAglB-L to a resolution of 1.90 Å (Figure ). The asymmetric unit contained one protein molecule. The calculated solvent content was 44.1% (VM = 2.20 Å3 Da-1). Data collection and refinement statistics are summarized in Table . The atomic coordinates of MBP-sAglB have been deposited in the Protein Data Bank, with the accession code 3WAI.The figures were generated with the PyMOL Molecular Graphics System, Version 1.3, (Schrödinger, LLC). The multiple sequence alignment was performed with the program MAFFT[]. […]

Pipeline specifications

Software tools Coot, PHENIX, PyMOL, MAFFT
Applications Small-angle scattering, Protein structure analysis
Organisms Archaeoglobus fulgidus