In March 2014, a symposium was held at Aarhus University to commemorate the 40th anniversary of the founding of its Division of Biostructural Chemistry by Professor Brian Clark. This article is a lightly edited version of the talk the author gave to open that event. It consists of some reflections about the discipline known as structural biology, and some comments about the author’s interactions with Professor Clark and the Division of Biostructural Chemistry over the years.

From ∼1970 to 2000, the year its crystal structure was solved, no biological structure was investigated more intensely than the ribosome. Some of the structural experiments done in the precrystallographic era produced information that significantly advanced our understanding of that enzyme. Other approaches yielded little that would be regarded as useful today, even if crystal structures had never been obtained. That experience is reviewed here to provide guidance for those faced with similar challenges in the future.

Structures have been obtained for the complexes that tiamulin, homoharringtonine, and bruceantin form with the large ribosomal subunit of Haloarcula marismortui at resolutions ranging from 2.65 to 3.2 Å. They show that all these inhibitors block protein synthesis by competing with the amino acid side chains of incoming aminoacyl-tRNAs for binding in the A-site cleft in the peptidyl-transferase center, which is universally conserved. In addition, these structures support the hypothesis that the species specificity exhibited by the A-site cleft inhibitors is determined by the interactions they make, or fail to make, with a single nucleotide, U2504 (Escherichia coli). In the ribosome, the position of U2504 is controlled by its interactions with neighboring nucleotides, whose identities vary among kingdoms.

Base pairing between the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs) and sequences in other eukaryotic RNAs target specific uridines for pseudouridylation. An RNA called HJ1 has been developed that interacts with the rRNA sequence targeted by the 5′ pseudouridylation pocket of human U65 snoRNA the same way as intact U65 snoRNA. Sequences on both strands of the analog of the U65 snoRNP pseudouridylation pocket in HJ1 pair with its substrate sequence, and the resulting complex, called HJ3, is strongly stabilized by Mg2+. The solution structure of HJ3 reveals an Ω-shaped RNA interaction motif that has not previously been described, which is likely to be common to all box H/ACA snoRNP-substrate complexes. The topology of the complex explains why the access of substrate sequences to snoRNPs is facile and how uridine selection may occur when these complexes form.

The quality of many macromolecular crystal structures published recently has been enhanced through the use of new methods for treating the effects of molecular motion and disorder on diffraction patterns, among them a technique called translation, libration, screw-axis (TLS) parameterization. TLS parameterization rationalizes those effects in terms of domain-scale, rigid-body motions and, interestingly, the models for molecular motion that emerge when macromolecular diffraction data are analyzed this way often make sense biochemically. Here it is pointed out that all such models should be treated with caution until it is shown that they are consistent with the diffuse scatter produced by the crystals that provided the diffraction data from which they derive.

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.