Computational protocol: The Crystal Structure of the Intact E. coli RelBE Toxin-Antitoxin Complex Provides the Structural Basis for Conditional Cooperativity

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Protocol publication

[…] Hexagonal crystals containing the RelB2E2 complex grew at 4°C in 1+1 μl sitting-drop vapor diffusion drops with a reservoir of 1.6−2.0 M ammonium sulfate and 0.1 M Na acetate, pH 4.6. Cryoprotection was achieved by gradual transfer of the crystals into 20% glycerol and heavy atom soaks prepared by addition of small amounts of heavy atom salts to the cryo solution. Native data and data from HA-soaked crystals were collected at the MAX-Lab (Lund, Sweden) and processed using XDS () for the derivative data set and Xia2 () for the native set. HA positions were initially located using RANTAN (), and an improved density-modified MIRAS map was subsequently obtained using only the Pt and Hg derivatives in SHARP (). Refinement was carried out by iterative model building in Phenix () and Coot () to a final R (Rfree) of 25.3% (28.5%; see for details). [...] Full-length E. coli RelER81A was expressed, purified, and crystallized as described previously (). Closer inspection of the crystallization drops revealed that they contained two morphologically different, three-dimensional crystal forms, and native data sets were collected from both types. For the previously described crystal form, belonging to the space group P21 (P1211) with three molecules per ASU, improved data extending to 1.8 Å were obtained at beamline ID29 of the European Synchrotron Radiation Facility (ESRF). The other crystal form turned out to belong to space group P212121 with two RelE molcules per ASU, and for this form, native data were collected at beamline X12 of the European Molecular Biology Laboratory-Deutsches Elektronen Synchrotron (EMBL-DESY) to a maximum resolution of 2.4 Å (). All data sets were processed using XDS () or Mosflm via Xia2 () and the structures were solved by molecular replacement in Phenix/Phaser (; ) using a search model derived from the published crystal structure of monomeric RelE (PDB ID 3KHA) (). From the map generated by Phaser, the models were fitted and rebuilt to include the C-terminal helix by iterative refinement in Phenix and rebuilding in Coot. The final R (Rfree) was 18.4% (21.9%) for the P1211 form and 23.6% (28.2%) for the P212121 form (see and for details). […]

Pipeline specifications

Software tools XDS, xia2, PHENIX, Coot, iMosflm
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Antitoxins, Estradiol