Computational protocol: Structural basis for ribosomal 16S rRNA cleavage by the cytotoxic domain of colicin E3

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Protocol publication

[…] The final data set was collected from two regions of a single crystal at beamline ID14-4 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Data were integrated and scaled using XDS. A starting model consisting of the empty 70S ribosome (without tRNAs, mRNA and ions) was used for initial refinement and phase calculation using CNS. The refinement process involved one cycle of rigid body (60 steps) refinement for each of the two 70S molecules in the asymmetric unit, one cycle of minimization (200 steps) and individual and grouped B-factor refinement. Unbiased mFo-DFc difference electron density maps calculated after initial refinement using a complete model of the ribosome, but without mRNA, tRNas or factor allowed modeling of mRNA, P-site tRNAfMet, non-cognate E-site tRNA and E3-rRNAse (E3-rRNase model, PDB code: 1E44). The final model included the entire P-site tRNAfMet, non-cognate E-site tRNA, mRNA (nucleosides 10 to 25) and the E3-RNase residues 0-96 (equal to registry 455-551 of full length colicin E3). The E3-RNase construct used in this study contained seven more residues at the N-terminal end (MEKNKPR). However no density could be observed for these residues indicating that this N-terminus makes no contacts with the ribosome and is rather flexible. Comparison of the 70S structure reported here with previously reported structures, showed r.m.s.d values of ~1.2 Å and ~0.4 Å for 16S and 23S rRNA (derived by least square fit of the phosphate backbones), respectively. The overall low r.m.s.d. values suggest that E3-rRNase binding and cleavage do not induce major global changes in the ribosome. Nevertheless, the higher r.m.s.d. value for the small subunit compared to the large subunit indicates that the 30S undergoes more pronounced conformational changes upon binding and cleavage by E3-RNase. The small subunit of the 70S–E3-rRNase structure reported here adopts an open form similar to what has been observed for native 30S subunits, but distinct to 30S or 70S structures with cognate tRNAs bound to the A-site,,,. It should be noted, that only one of the E3-RNase molecules in the asymmetric unit could be fully modeled, although with elevated B-factor (average B-factor ~146) compared to the surrounding environment, while the other E3-rRNase molecule could be build (with occupancy = 1) only from residue 1 to 14 due to lack of clear electron density, showing that the small subunit in the open form shows a significant level of conformational variability (especially in the shoulder region). The 16S rRNA in both ribosome molecules, however was cleaved. The N-terminal end of S13 (residues 119-125) was removed due to a lack in density. L31 that previously contained model and registry ambiguity was corrected referring to the Genbank registry and was built to contain residue 1 to residue 57 according to the unbiased map. The final refinement in CNS had Rwork/Rfree of 22.8%/27.0%. Data and refinement statistics are reported in . […]

Pipeline specifications

Software tools XDS, CNS
Applications Small-angle scattering, Protein structure analysis
Chemicals Etodolac