Acta Crystallographica. Section D, Structural Biology最新文献

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

The axoneme, a microtubule-based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo-EM and cryo-ET have allowed a molecular-level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo-EM and cryo-ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI-guided tools that are transforming structural biology.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.

The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO₂ and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.