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

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.

The use of polyethylene glycol in the crystallization of biological macromolecules and its appearance in the resulting crystals is discussed.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential tool in structural biology. However, automating repetitive tasks remains an ongoing challenge in cryo-EM data-set processing. Here, we present a platform-independent convolutional neural network (CNN) tool for assessing the quality of 2D averages to enable the automatic selection of suitable particles for high-resolution reconstructions, termed CryoSift. We integrate CryoSift into a fully automated processing pipeline using the existing cryosparc-tools library. Our integrated and customizable 2D assessment workflow enables high-throughput processing that accommodates experienced to novice cryo-EM users.