Computational protocol: A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys

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Protocol publication

[…] The single-wavelength anomalous dispersion (SAD) data sets from the SeMet derivative protein co-crystals with GMPPNP or GDP were collected at beamline BL5A of the Photon Factory (Tsukuba, Japan). The data set of the native protein was collected at beamline BL41XU of SPring-8 (Harima, Japan). All data were processed using the HKL2000 program suite []. The MM1309 crystals belong to the orthorhombic space group P21212, with unit cell dimensions of a = 62.06, b = 108.7, c = 58.32 Å, and the asymmetric unit contains one MM1309 molecule. The selenium sites were identified using SnB [] with the SeMet/GMPPNP data set. The selenium sites were refined and the initial phases were calculated with SOLVE []. The phases were improved with density modification, using RESOLVE []. The initial model was automatically built by RESOLVE and ArpWarp [], and was manually refined using O [], CueMol [http://cuemol.sourceforge.ge.jp/en], and Coot []. The atomic model was refined using CNS [], REFMAC5 [], and PHENIX []. The models showed good stereochemistry and geometry, as analyzed by the programs Procheck [] and Molprobity [http://molprobity.biochem.duke.edu/, ]. The structures of the GDP-bound and apo forms were solved by the molecular replacement method, using Molrep [] with the GMPPNP-bound form model as the search model, and refined in the same manner as the GMPPNP-bound form. Graphical images were prepared with the program PyMOL [http://pymol.sourceforge.net/]. All data collection and refinement statistics are summarized in Table . Superimpositions of the Cα traces of the MM1309 structures were produced by the program secondary structure matching (SSM) []. […]

Pipeline specifications

Software tools CueMol, Coot, REFMAC5, PHENIX, PROCHECK, MolProbity, Molrep, PyMOL
Application Protein structure analysis
Organisms Methanosarcina mazei, Methanosarcina barkeri
Chemicals Guanosine Diphosphate, Guanosine Triphosphate