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

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.

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)₈-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^α 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)₄, with a lower binding energy of −6.3 kcal mol⁻¹. 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.

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 P2₁2₁2₁ (including one previously reported, PDB entry 9unt) and three in the tetragonal P4₁2₁2. Despite large variations in pH, salt and precipitant, all structures were nearly identical, with pairwise C^α r.m.s.d.s of 0.06–0.33 Å. Structures within the same space group were indistinguishable, with pairwise C^α 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.

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 S_Ni 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.