Developing NMR methods for macromolecular machines: Measurement of residual dipolar couplings to probe dynamical regions of the ribosome

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamical regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the stalk protein bL12, and this direct structural information was used to refine a 3D structure of its C-terminal domain (CTD). Orientational constraints on the CTD alignment arising from the semiflexible linker were further probed by a paramagnetic alignment strategy, and provided direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes.