This case report describes single surface substitutions that improve the crystallizability and diffraction properties of a flexible two-domain protein. InlB comprises the internalin domain and the B repeat of the Listeria monocytogenes invasion protein InlB. The InlB 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 InlB 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 InlB. 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.

Plant chitinases are found in different organs such as stems, seeds, flowers, corms, tubers and bulbs. In this study, we report the crystal structure of the class IIIb chitinase from Crocus vernus (L.) Hill corms and a thorough comparative analysis with other plant chitinases, especially focusing on molecular-docking interactions between the protein and ligands (allosamidin and chitin oligomer). The C. vernus chitinase (CvChi; PDB entry 3sim) structure has been refined to a crystallographic R factor of 15.5% at a resolution of 2.1 Å. The asymmetric unit is comprised of two chains with 550 residues and 406 water molecules. CvChi has a (β/α)-barrel fold and the catalytic residues of CvChi (Asp123, Asp125 and Glu127) are directly located in the cavity of the barrel. CvChi belongs to the GH18 chitinase family and showed 50% and 16% sequence identity to GH18 chitinases from the fern Pteris ryukyuensis (PrChiA-cat; PDB entry 4rl3) and Hevea brasiliensis (hevamine; PDB entry 1llo), respectively. Structural alignment of the C atoms of CvChi with PDB entries 7xmh, 4rl3 and 1llo showed r.m.s.d. values of 0.547, 0.897 and 3.8 Å, respectively. Interestingly, two loops (L2 and L3) important for sugar cleavage are larger in CvChi compared with both PDB entries 4rl3 and 1llo. The affinity of CvChi towards allosamidin is lower than those of other GH18 chitinases. Molecular docking revealed that several hydrogen bonds found in the crystal structure of the hevamine-allosamidin complex were missing in the modeled structure of the CvChi-allosamidin complex. The active residues DXDXE of CvChi form a hydrogen bond to allosamidin, compared with two hydrogen bonds in PrChiA-cat. However, CvChi exhibits higher affinity for the chitin oligomer (GlcNAc), with a lower binding energy of -6.3 kcal mol. Purified CvChi showed maximum endochitinase activity at concentrations of 500 and 1000 ng per assay. CvChi exhibited an antifungal effect against the phytopathogenic fungus Fusarium oxysporum at 500 µg per well, inhibiting about half of the fungal growth.

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.

Short-chain dehydrogenases (SDRs) are a family of NAD(P)-dependent enzymes involved in redox reactions, specifically carbonyl-alcohol reductions. Here, we report the apo and NAD-bound structures of an SDR from the pathogenic organism Brucella ovis. B. ovis primarily affects sheep and other livestock, resulting in reduced fertility. Based on sequence and structural alignment, the B. ovis SDR (BoSDR) is a classical SDR. Classical SDRs have a canonical YxxxK active-site sequence in which the catalytic general base is a tyrosine residue located at position 163. In addition, the putative active site also contains a serine residue (Ser150) and lysine residue (Lys167) that are hypothesized to be involved in catalysis. BoSDR is a biological and crystallographic tetramer. In the coenzyme-bound structure, two different orientations of the NAD coenzyme are fortuitously observed, which provides insights into the conformational changes that accompany coenzyme binding. The apo and NAD-bound structures provide valuable information about the unique structural features of enzymes in the SDR superfamily.

Pterin glycosides are widely distributed in cyanobacteria and have been implicated in the regulation of phototaxis and photosynthesis. Here, we identified a new uridine diphosphate glucose:tetrahydrobiopterin α-glucosyltransferase, termed PsBGluT, from Pseudanabaena sp. Chao 1811, which catalyzes the formation of pterin glycosides. We solved crystal structures of apo PsBGluT and its UDP-bound form at 2.8 and 2.3 Å resolution, respectively. PsBGluT forms a homodimer, with each subunit adopting a canonical GT-B fold composed of two Rossmann-like domains. Structural analysis combined with molecular docking revealed the binding sites for both the donor UDP-glucose and the acceptor tetrahydrobiopterin. Based on these findings, we proposed that PsBGluT operates via an Si retaining catalytic mechanism. This study advances our understanding of pteridine glycosylation and also provides a structural basis for investigating the photosynthetic signaling pathways in cyanobacteria.

Proteins crystallized under varied conditions typically exhibit nearly identical overall structures, with deviations confined to flexible loops or side-chain orientations. In this study, we report the extraordinary crystallization properties of PF1765 from Pyrococcus furiosus, which crystallized from the same batch of protein preparation in 104 of 192 different crystallization conditions. This yielded ten high-resolution structures (1.1-1.5 Å) across two space groups: seven in the orthorhombic space group P222 (including one previously reported, PDB entry 9unt) and three in the tetragonal P422. Despite large variations in pH, salt and precipitant, all structures were nearly identical, with pairwise C r.m.s.d.s of 0.06-0.33 Å. Structures within the same space group were indistinguishable, with pairwise C r.m.s.d.s of 0.06-0.09 and 0.09-0.14 Å for the tetragonal and orthorhombic space groups, respectively. These results confirm that the overall structure remained unaffected by the large chemical variability during crystallization. Consistently, major crystal contacts were conserved across the two space groups, while hydration mapping identified six conserved waters across all of the structures. Interestingly, rotameric differences were observed between space groups, where residues Ser6, Glu7, Pro17, Asn18, Pro41, Pro42, Val43 and Arg72 adopt distinct conformations reflecting lattice-specific packing. Collectively, PF1765 emerges as a hyper-crystallizable protein that provides a consistent framework for analyzing lattice-dependent microheterogeneity, packing and hydration-site conservation at atomic resolution. Its compact, rigid single-domain structure and reproducible crystallization behavior indicate potential use as a fusion domain to aid the crystallization of membrane proteins or complexes by promoting ordered lattice formation. However, this study does not examine crystal nucleation or growth kinetics under varying conditions, which remain important directions for future investigation.

Ketohexokinase (KHK) catalyses the initial step in fructose metabolism, converting the furanose form of D-fructose to fructose 1-phosphate in an ATP-dependent reaction. Given its central role in metabolic pathways, KHK has emerged as a target for pharmacological intervention in the treatment of non-alcoholic fatty liver disease, metabolic syndrome, type 2 diabetes and obesity. KHK exists as two isoforms, A and C, which arise from alternative splicing of exon 3, resulting in a differing 45-amino-acid sequence within the 298-amino-acid primary structure of the enzyme. KHK is a biological homodimer, with each subunit adopting an α/β-fold architecture that interlocks with a β-clasp domain. In the case of KHK-C at least two distinct conformations of the β-clasp domain have been identified, whereas this conformational flexibility had not been observed in KHK-A. Here, X-ray crystallographic structural investigations of unliganded murine KHK-A refined to 1.37 Å resolution revealed the adoption of two conformations similar to those adopted by the human ortholog, suggesting that this structural feature is conserved across species. The functional significance of these conformational changes in KHK-A is of particular interest as this isoform has been implicated in cancer metastasis through a `moonlighting' protein kinase activity. Understanding the mechanistic role of conformational shifts in KHK-A may provide insights into its broader physiological functions and therapeutic potential.

Fucoidan is a complex, sulfated polysaccharide primarily found in brown algae, where it plays important structural and protective roles. Due to its abundance in marine ecosystems, many marine bacteria have evolved diverse and specialized enzymatic systems to degrade fucoidan, although the functions and structures of many of these enzymes remain uncharacterized. Here, we describe the structure of a newly identified fucosidase, FucWf4, which cleaves terminal, unsulfated fucose residues from linear, sulfated fucoidan. FucWf4 does not belong to any known glycoside hydrolase (GH) family, but shows the greatest similarity to GH29 fucosidases. We present the first crystal structure of FucWf4 in complex with fucose, revealing a unique C-terminal domain that resembles a carbohydrate-binding module, although it may have lost its carbohydrate-binding capacity and is absent from canonical GH29 enzymes. Docking experiments suggest the presence of a -1 subsite containing a potential sulfate-binding pocket, which may underlie the substrate specificity of the enzyme. Furthermore, sequence analysis of FucWf4 homologs reveals two distinct clades, likely corresponding to functionally divergent groups. Together, these findings provide new insights into the molecular basis of fucoidan recognition and degradation by this novel enzyme subfamily, laying the groundwork for future functional and structural studies.

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide into hydrogen peroxide and molecular oxygen, serving as a key defence against oxidative damage. Despite extensive studies, the full structural characterization of HO-binding sites in MnSOD remains largely unexplored. Previous HO-soaked MnSOD structures have identified two distinct HO-binding sites: one directly ligated to the catalytic manganese (LIG position) and another at the active-site gateway (PEO position) between the second-shell residues Tyr34 and His30. In this study, a kinetically impaired Gln143Asn MnSOD variant is used to trap and explore additional HO-binding sites beyond the second-shell solvent gate. In the wild-type enzyme, Gln143 mediates proton transfers with the manganese-bound solvent (WAT1) to drive redox cycling of the metal, which is necessary for effective superoxide dismutation. Substitution with Asn stalls catalysis because the increased distance from WAT1 disrupts critical proton-coupled electron-transfer (PCET) events, and the redox cycling of the active-site metal is impaired. This, in turn, stalls the electrostatic cycling of positive charge on the enzyme surface and enhances the likelihood of trapping transient HO-bound states in this variant. The results reveal several HO molecules leading up to the active site, in addition to the canonical LIG and PEO positions.

We describe a device and a method for changing the ambient solution of a macromolecular crystal. The approach is gentle, automated, inexpensive and open source. Examples are given of the equilibration of three different crystals to new solutions with exchange times ranging from 5 to 180 min. In each case direct transfer of the crystal to the new solution causes cracking, which is eliminated with gradient equilibration using the described device. Crystals equilibrated with the device produce high-quality diffraction that yields refined structures comparable to those determined previously. The device offers a more systematic and labor-saving workflow than current practice both for performing diffraction analysis of macromolecular crystals and for investigating the response of macromolecular crystals to changes in solution composition.

Gasdermin D (GSDMD) is a protein that has gained significant attention in recent years due to its crucial role in inflammatory cell death, particularly pyroptosis. Pyroptosis is a highly inflammatory form of programmed cell death that is triggered by various microbial infections and sterile inflammatory stimuli. GSDMD acts as an executioner molecule in this process, leading to the release of pro-inflammatory cytokines and amplifying the immune response. Here, we present a higher resolution, significantly improved apo crystal structure of the deposited mouse structure model that will be beneficial for structure-based drug-design approaches towards this important pharmacological target.

Ease of access to data, tools and models expedites scientific research. In structural biology there are now numerous open repositories of experimental and simulated data sets. Being able to easily access and utilize these is crucial to allow researchers to make optimal use of their research effort. The tools presented here are useful for collating existing public cryoEM data sets and/or creating new synthetic cryoEM data sets to aid the development of novel data processing and interpretation algorithms. In recent years, structural biology has seen the development of a multitude of machine-learning-based algorithms to aid numerous steps in the processing and reconstruction of experimental data sets and the use of these approaches has become widespread. Developing such techniques in structural biology requires access to large data sets, which can be cumbersome to curate and unwieldy to make use of. In this paper, we present a suite of Python software packages, which we collectively refer to as PERC (profet, EMPIARreader and CAKED). These are designed to reduce the burden which data curation places upon structural biology research. The protein structure fetcher (profet) package allows users to conveniently download and cleave sequences or structures from the Protein Data Bank or AlphaFold databases. EMPIARreader allows lazy loading of Electron Microscopy Public Image Archive data sets in a machine-learning-compatible structure. The Class Aggregator for Key Electron-microscopy Data (CAKED) package is designed to seamlessly facilitate the training of machine-learning models on electron microscopy data, including electron-cryo-microscopy-specific data augmentation and labeling. These packages may be utilized independently or as building blocks in workflows. All are available in open-source repositories and designed to be easily extensible to facilitate more advanced workflows if required.

Several cyanobacterial species, including Crocosphaera subtropica ATCC 51142, accumulate cyanobacterial starch instead of glycogen, although nearly all cyanobacteria accumulate glycogen. The glycogen-producing Synechococcus elongatus PCC 7942 possesses one α-glucan phosphorylase (Pho) isozyme, whereas strain 51142 has three Pho isozymes. Based on their primary structures, these enzymes belong to glycosyl transferase (GT) family 35, with the cyanobacterial GT35-type Phos further subdivided into types I-III. In this study, to elucidate the significance of the coexistence of multiple GT35-type Pho isozymes, those from strain 51142 (type I, cce_1629; type II, cce_1603 and cce_5186) and strain 7942 (type I, Synpcc7942_0244) were overexpressed in Escherichia coli and biochemically characterized. All isozymes catalysed the phosphorolysis and reverse phosphorolysis reactions. The type I isozyme from a cyanobacterial starch-producing strain (cce_1629) differed in substrate specificity and specific activity compared with the others. The behaviour towards the effectors (AMP and ATP) of the type I and type II isozymes differed from each other. These findings enhance our understanding of the roles of cyanobacterial Pho isozymes in α-glucan metabolism. Furthermore, recombinant cce_1603 was crystallized using the hanging-drop vapour-diffusion method. Crystals were obtained at 293 K in the presence of 10 mM maltoheptaose, 45%(w/v) PEG 400, 0.1 M Tris-HCl pH 8.0, 0.2 M lithium sulfate. The crystals belonged to space group R32 (hexagonal setting) with unit-cell parameters a = b = 267.23, c = 204.43 Å, and diffracted to beyond 2.70 Å resolution. Matthews coefficient calculations indicated the presence of two molecules in the asymmetric unit. Structural determination is currently under way. The crystal structure of cce_1603 will aid in the understanding of the structural basis of cyanobacterial GT35-type Pho isozymes.

Bacillus subtilis DegQ is a 46-amino-acid regulatory protein involved in the DegS-DegU two-component system. DegQ promotes the phosphorylation of DegU by DegS, switching the function of DegU from competence to the induction of poly-γ-glutamate production. To elucidate its structural role, we determined the crystal structures of wild-type DegQ and its mutant DegQS25L. Each DegQ monomer folds into a single α-helix, and four monomers assemble into a tetramer characterized by a four-helix coiled-coil structure. Within the tetramer, two adjacent helices are oriented in the same direction, while the other two are oriented oppositely, forming a pseudo-twofold symmetric arrangement. The mutant form displays disrupted symmetry due to altered helix packing, which is caused by shifts in the coiled-coil heptad register induced by the mutation. Structural predictions using AlphaFold3 suggest that DegQ likely binds to the N-terminal helix bundle of DegS, either as a dimer or as individual monomers. These findings provide structural insight into DegQ oligomerization and its potential role in modulating DegS autophosphorylation and DegU binding.

The focused issue on the SAMPREP workshop is introduced. The virtual issue is available at https://journals.iucr.org/special_issues/2025/samprep23/.

The enzyme D-aspartate oxidase (DDO) oxidizes acidic D-amino acids using the coenzyme flavin adenine dinucleotide to generate the corresponding α-keto acids and ammonia. DDO differs from D-amino-acid oxidase (DAAO), which acts on neutral and basic D-amino acids. Although the enzymatic properties of DDO have been characterized in several species, the structure of DDO had remained unclear. The structure of DDO derived from Cryptococcus humicola strain UJ1 (chDDO) was determined by X-ray crystallography at 1.70 Å resolution. While the three-dimensional structures of DAAOs are known to be homodimers, chDDO forms a homotetramer. This difference was found to be caused by the deletion of one loop and the insertion of two loops.

Virulence protein J (VirJ) is a periplasmic protein encoded by the bacterial pathogen Brucella abortus and is important for its virulence. The VirJ homologue AcvB from Agrobacterium tumefaciens was found to be a lysyl-phosphatidylglycerol hydrolase that contains two domains, D1 and D2. Interestingly, both VirJ and AcvB are associated with the type IV secretion system (T4SS) activity in the respective bacteria. To date, no structural information is available for these proteins, limiting our understanding of their function. Here, we have purified, crystallized and determined the crystal structure of the N-terminal domain 1 of VirJ (VirJ) at a resolution of 1.7 Å. Our structural analysis shows that VirJ adopts an α/β-hydrolase fold but lacks the characteristic catalytic triad. The structure presented here may help to decipher the function of VirJ in Brucella spp. and other bacterial pathogens, as well as its contribution to the T4SS function.

The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.

Histone deacetylase inhibitors (HDACi) are widely used in cancer therapy but often suffer from off-target effects due to their pan-inhibitory activity towards zinc-dependent enzymes. Vorinostat (SAHA), a hydroxamate-based HDACi, has been shown to lack isoform selectivity, potentially leading to unintended interactions with other metalloenzymes. Here, we report high-resolution crystal structures of SAHA bound to human carbonic anhydrase II (CA II) and a carbonic anhydrase IX (CA IX) active-site mimic. Structures determined at room temperature and 100 K revealed two distinct SAHA conformers in both CA II and the CA IX mimic, with the hydroxamate moiety displacing the zinc-bound water and adopting either a tetrahedral or pentahedral coordination to Zn. Differences in hydrophobic interactions were observed between CA II and the CA IX mimic due to the F131V amino-acid difference between the two enzymes. SwissDock modeling accurately predicted the SAHA binding orientations observed in crystallography. Thermal shift assays using nanoDSF showed minimal stabilization of either CA by SAHA, in contrast to the potent CA inhibitor acetazolamide. Binding-energy calculations suggest that SAHA may bind carbonic anhydrases with affinities comparable to its HDAC targets. These findings highlight potential off-target binding of SAHA to carbonic anhydrases, which may contribute to its clinical side effects. The results also suggest that hydroxamates may serve as a nonsulfonamide scaffold for novel CA inhibitors, although isoform selectivity remains a challenge.

The α/β-hydrolase fold superfamily includes esterases and hydroxynitrile lyases which, despite catalyzing different reactions, share a Ser-His-Asp catalytic triad. We report a 1.99 Å resolution crystal structure of HNL6V, an engineered variant of hydroxynitrile lyase from Hevea brasiliensis (HbHNL) containing seven amino-acid substitutions (T11G, E79H, C81L, H103V, N104A, G176S and K236M). The structure reveals that HNL6V maintains the characteristic α/β-hydrolase fold while exhibiting systematic shifts in backbone and catalytic atom positions. Compared with wild-type HbHNL, the C positions in HNL6V differ by a mean of 0.2 ± 0.1 Å, representing a statistically significant displacement. Importantly, the catalytic triad and oxyanion-hole atoms have moved 0.2-0.8 Å closer to their corresponding positions in SABP2, although they remain 0.3-1.1 Å from fully achieving the configuration of SABP2. The substitutions also increase local flexibility, particularly in the lid domain covering the active site. This structural characterization demonstrates that targeted amino-acid substitutions can systematically shift catalytic geometries towards those of evolutionarily related enzymes.