Acta crystallographica. Section F, Structural biology communications最新文献

The XPD helicase plays a critical role in DNA repair and serves as a model for structural studies of superfamily 2 (SF2) helicases. We report a novel orthorhombic crystal form of DNA-free Thermoplasma acidophilum XPD (TaXPD) obtained under high ionic strength conditions generated by sodium potassium tartrate and NaCl-based vapor-diffusion conditions, in contrast to earlier previously reported conditions that used polyols (PEG) or diols (MPD). The crystals belonged to space group P2₁2₁2₁ (a = 59.53, b = 96.00, c = 159.09 Å) and diffracted to 2.13 Å resolution, yielding the highest resolution TaXPD structure to date. Structural analysis showed that this crystal form contains fewer intermolecular interfaces than the previously reported hexagonal lattice, as supported by Protein Interfaces, Surfaces and Assemblies (PISA) analysis. This supports the determination of the 510-514 loop in the long-form DNA-free TaXPD, which was previously disordered in other structures. This work highlights how crystallization conditions influence lattice organization, structural completeness and diffraction quality. In this structure, Tyr425 adopts a conformation that may regulate DNA access in the DNA-free state.

Despite the theoretical advantages of phosphorus single-wavelength anomalous diffraction (P-SAD) for nucleic acid phasing, its application remains limited due to high atomic displacement parameters and an unfavourable ratio of unique reflections to anomalous scatterers. In this study, we report the crystal structure of an RNA complex composed of four strands, which was solved by experimental phasing after AlphaFold3 failed to produce reliable models. Bromine single-wavelength anomalous diffraction (Br-SAD) data were collected at 0.916 Å on beamline I04 at Diamond Light Source, while phosphorus anomalous data were obtained at 3.024 Å on beamline I23. The structure was successfully phased using bromine anomalous scattering, and phosphorus anomalous peaks corroborated the backbone positions and validated the model. Attempts to phase the structure directly from phosphorus data failed, consistent with theoretical predictions that successful SAD phasing requires a significantly higher reflection-to-scatterer ratio. The final models reveal an RNA complex stabilized by Watson-Crick and Hoogsteen base pairing, forming a pseudo-helical complex instead of the anticipated hairpin stem-loop, likely reflecting crystallization artefacts. This work demonstrates the complementary use of bromine and phosphorus anomalous signals in RNA crystallography.

The striking blue hue of live Homarus americanus, known as the American lobster, arises from the interaction of astaxanthin with the carotenoprotein α-crustacyanin. The mechanism underlying the bathochromic shift of the chromophore from its unbound red form (λ_max = 472 nm) to the blue protein-bound form (λ_max = 631 nm) is the subject of attention from the food and nutraceutical industries for the development of versatile food colourants. Here, we present sample purification and characterization of the α- and β-crustacyanin pigments from the American lobster for crystallographic studies and cryo-EM. Moving from H. gammarus to H. americanus for complex isolation, together with an integrated biophysical characterization, resulted in the production, for the first time, of α-crustacyanin crystals that diffracted to 6.3 Å resolution at ⟨I/σ(I)⟩ = 1.0 and high-quality negative-stain and cryo-EM images.

Here, we report the X-ray structural analysis of dihydroorotase from Methanococcus jannaschii co-crystallized with dihydroorotate at pH 6.5. The crystals are isomorphous with the crystals of the apoenzyme, with space group P3₂21 and unit-cell dimensions a = b = 111.4, c = 101.2 Å. The structure was refined to R = 0.169 and R_free = 0.186 at a resolution of 1.87 Å at room temperature. During crystallization the degradative reaction took place, and the electron density in the active site corresponds to the substrate carbamoyl aspartate. Carbamoyl aspartate interacts with the protein in the active site in a manner similar to that observed for Escherichia coli and human dihydroorotases. However, the flexible loop (residues 140-151) adopts both conformations in the crystal, loop-out and loop-in, with the loop-out conformation having higher occupancy. This contrasts with our expectations for the flexible loop to be exclusively in the loop-in conformation, which is the conformation that it adopts to stabilize the binding of the substrate in the known systems. Additional studies with substrate analogs that resemble carbamoyl aspartate and different crystallization conditions would provide further insight into the conformation of the flexible loop in this system.

This case report describes single surface substitutions that improve the crystallizability and diffraction properties of a flexible two-domain protein. InlB₃₉₂ comprises the internalin domain and the B repeat of the Listeria monocytogenes invasion protein InlB. The InlB₃₉₂ wild type yielded very few poorly reproducible hits in crystallization screens and the crystals had a diffraction limit of worse than 3.0 Å. It seems reasonable to assume that this crystallization bottleneck is caused by interdomain flexibility, given that crystals of the isolated internalin domain or B repeat diffract to high resolution. A previously identified variant, T332E, showed improved crystallization and diffraction. Here, two additional InlB₃₉₂ variants are described with single threonine-to-tyrosine or valine-to-glutamate substitutions that produced crystals directly in initial screens and, without optimization, diffracted to 1.6 and 1.45 Å resolution, respectively. The mutated residues do not participate in intramolecular interdomain interactions but mediate crystal contacts, indicating that specific surface properties, rather than interdomain flexibility per se, impede the crystallization of wild-type InlB₃₉₂. Notably, the beneficial glutamate substitutions contrast with the generally recognized underrepresentation of glutamate in crystal contacts and the high entropic cost of fixing an otherwise flexible side chain with many rotatable bonds in a crystal contact. The reported results suggest that surface mutations can help crystallization even if they increase the entropy of the respective residue. More broadly, the observations are consistent with the hypothesis that negative evolutionary design limits fortuitous lattice formation of proteins and the resulting expectation that random mutations of surface residues are likely to improve crystallizability.

The name of one of the authors in the article by Sharma et al. [(2023), Acta Cryst. F79, 224-230] is corrected.

The adapter proteins of the 14-3-3 family modulate the activity and/or localization of their binding partners, which they capture if a generic target motif is in its phosphorylated state. Here, we report the identification of potential 14-3-3 binding sites in human sirtuin deacylases by bioinformatic analysis. We then characterize the interactions of peptides representing phosphorylation sites in sirtuin 3 (pS103) and sirtuin 1 (pS670) with 14-3-3 proteins. We further describe the crystal structures of complexes of 14-3-3σ with either of the two phosphopeptides. As a conclusion, we propose a more extended 14-3-3 binding mode on the N-terminal side of the phosphorylation site and the possibility of nongeneric motifs and conformations on the C-terminal side, still resulting in the known high binding affinity of the two partners.