Acta Crystallographica. Section D, Structural Biology最新文献

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.

Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.

Five new Co-editors are appointed to the Editorial Board of Acta Cryst. D - Structural Biology.

The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) - F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H₂O₂) into molecular oxygen and water. In all monofunctional catalases the pathway that H₂O₂ takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H₂O₂ routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with k_cat and k_cat/K_m values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P2₁2₁2 enabled the successful modeling of 96% of residues in the asymmetric unit, with final R_work and R_free values of 0.199 and 0.229, respectively.

The Guest Editors provide an introduction to the special issue of articles based on talks at the CCP4 Study Weekend 2022, which is available at https://journals.iucr.org/special_issues/2023/CCP42022/.