Ribosomal protein L9: a structure determination by the combined use of X-ray crystallography and NMR spectroscopy.

The structure of protein L9 from the Bacillus stearothernophilus ribosome has been determined at 2.5 A resolution by refinement against single crystal X-ray diffraction data with additional constraints provided by NMR data. This highly elongated protein consists of two domains separated by a nine-turn connecting helix. Conserved aromatic and positively charged amino acid residues on the surface of each domain are likely to be directly involved in binding 23 S ribosomal RNA. The shape of the protein, with its two widely spaced RNA-binding sites, suggests that it may serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of the ribosomal RNA. The combined use of X-ray and NMR data in the refinement procedure was essential in defining the N-terminal domain of the protein, which was relatively poorly determined by the X-ray data alone. In addition to resolving the ambiguities in defining the hydrophobic core and side-chain conformations with the N-terminal domain, this combined NMR-X-ray analysis provides the first detailed and accurate view of the N-terminal RNA-binding site. NMR data also showed that the N-terminal domain is stable in solution, indicated by amide protons that are protected from solvent exchange. The lack of definition of the N-terminal domain in the X-ray structure is therefore likely due to packing disorder within the crystal rather than structural instability. This combined NMR-X-ray analysis provides a useful model as to how X-ray and NMR data can be practically and logically combined in the determination of the structure of a single protein molecule.