Acta Crystallographica. Section D, Structural Biology最新文献

Histidine can be protonated on either or both of the two N atoms of the imidazole moiety. Each of the three possible forms occurs as a result of the stereochemical environment of the histidine side chain. In an atomic model, comparing the possible protonation states in situ, looking at possible hydrogen bonding and metal coordination, it is possible to predict which is most likely to be correct. A more direct method is described that uses quantum-mechanical methods to calculate, also in situ, the minimum geometry and energy for comparison, and therefore to more accurately identify the most likely protonation state.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α₂β₂ tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

Serial crystallography, born from groundbreaking experiments at the Linac Coherent Light Source in 2009, has evolved into a pivotal technique in structural biology. Initially pioneered at X-ray free-electron laser facilities, it has now expanded to synchrotron-radiation facilities globally, with dedicated experimental stations enhancing its accessibility. This review gives an overview of current developments in serial crystallography, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings.

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced. Tested on endothiapepsin crystals with four soaked fragments, thaumatin and SARS-CoV-2 3CL^pro, Cryo2RT reveals unique ligand-binding poses, offers a comparable throughput to cryo-crystallography and eases the exploration of structural dynamics at various temperatures.

Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example.

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It bears a significant global health burden with limited treatment options, thus calling for the development of new and effective drugs. Certain trypanosomal metabolic enzymes have been suggested to be druggable and valid for subsequent inhibition. In this study, the crystal structure of glycerol kinase from T. cruzi, a key enzyme in glycerol metabolism in this parasite, is presented. Structural analysis allowed a detailed description of the glycerol binding pocket, while comparative assessment pinpointed a potential regulatory site which may serve as a target for selective inhibition. These findings advance the understanding of glycerol metabolism in eukaryotes and provide a solid basis for the future treatment of Chagas disease.

The interpretation of cryo-EM maps often includes the docking of known or predicted structures of the components, which is particularly useful when the map resolution is worse than 4 Å. Although it can be effective to search the entire map to find the best placement of a component, the process can be slow when the maps are large. However, frequently there is a well-founded hypothesis about where particular components are located. In such cases, a local search using a map subvolume will be much faster because the search volume is smaller, and more sensitive because optimizing the search volume for the rotation-search step enhances the signal to noise. A Fourier-space likelihood-based local search approach, based on the previously published em_placement software, has been implemented in the new emplace_local program. Tests confirm that the local search approach enhances the speed and sensitivity of the computations. An interactive graphical interface in the ChimeraX molecular-graphics program provides a convenient way to set up and evaluate docking calculations, particularly in defining the part of the map into which the components should be placed.

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

The determination of the atomic resolution structure of biomacromolecules is essential for understanding details of their function. Traditionally, such a structure determination has been performed with crystallographic or nuclear resonance methods, but during the last decade, cryogenic transmission electron microscopy (cryo-TEM) has become an equally important tool. As the blotting and flash-freezing of the samples can induce conformational changes, external validation tools are required to ensure that the vitrified samples are representative of the solution. Although many validation tools have already been developed, most of them rely on fully resolved atomic models, which prevents early screening of the cryo-TEM maps. Here, a novel and automated method for performing such a validation utilizing small-angle X-ray scattering measurements, publicly available through the new software package AUSAXS, is introduced and implemented. The method has been tested on both simulated and experimental data, where it was shown to work remarkably well as a validation tool. The method provides a dummy atomic model derived from the EM map which best represents the solution structure.