Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling

The Escherichia coli ribosomal protein L7/L12 is central to the translocati on step of translation, and it is known to be flexible under some condition s. The assignment of electron density to L7/L12 was not possible in the rec ent 2.4 Angstrom resolution x-ray crystallographic structure (Ban, N,, Niss en, P,, Hansen, J,, Moore, P, B,, and Steitz, T, A. (2000) Science 289, 905 -920), We have localized the two dimers of L7/L12 within the structure of t he 70 S ribosome using two reconstitution approaches together with cryo-ele ctron microscopy and single particle reconstruction. First, the structures were determined for ribosomal cores from which protein L7/L12 had been remo ved by treatment with NH4Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mappi ng revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11. Second, ribosomes were reconstitut ed using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 Angs trom gold derivative. The reconstruction from cryo-electron microscopy imag es and difference mapping placed the gold at four interfacial positions. Th e finding of multiple sites for the C-terminal domain of L7/L12 suggests th at the conformation of this protein may change during the steps of elongati on and translocation.