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Preview: Acta Crystallographica Section F

Acta Crystallographica Section F

Acta Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structur

Published: 2017-10-01


Crystal structure of type II NADH:quinone oxidoreductase from Caldalkalibacillus thermarum with an improved resolution of 2.15 Å


Type II NADH:quinone oxidoreductase (NDH-2) is a respiratory enzyme found in the electron-transport chain of many species, with the exception of mammals. It is a 40–70 kDa single-subunit monotopic membrane protein that catalyses the oxidation of NADH and the reduction of quinone molecules via the cofactor FAD. NDH-2 is a promising new target for drug development given its essential role in many bacterial species and intracellular parasites. Only two bacterial NDH-2 structures have been reported and these structures are at moderate resolution (2.3–2.5 Å). In this communication, a new crystallization platform is reported that produced high-quality NDH-2 crystals that diffracted to high resolution (2.15 Å). The high-resolution NDH-2 structure was used for in silico quinone substrate-docking studies to investigate the binding poses of menadione and ubiquinone molecules. These studies revealed that a very limited number of molecular interactions occur at the quinone-binding site of NDH-2. Given that the conformation of the active site is well defined, this high-resolution structure is potentially suitable for in silico inhibitor-compound screening and ligand-docking applications.

Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger


Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase from Aspergillus niger is a small globular protein containing a disulfide bond. The structure of A. niger SBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from an Escherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Å resolution. The SBD consisted of seven β-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each β-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the Cα atom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, the B factors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

Large-scale crystallization and neutron crystallographic analysis of HSP70 in complex with ADP


HSP70 belongs to the heat-shock protein family and binds to unfolded proteins, driven by ATP hydrolysis, in order to prevent aggregation. Previous X-ray crystallographic analyses of HSP70 have shown that HSP70 binds to ADP with internal water molecules. In order to elucidate the role of the water molecules, including their H/D atoms, a neutron diffraction study of the human HSP70 ATPase domain was initiated. Deuterated large crystals of the HSP–ADP complex (1.2–1.8 mm3) were successfully grown by large-scale crystallization, and a neutron diffraction experiment at BIODIFF resulted in diffraction to a maximum resolution of 2.2 Å. After data reduction, the overall completeness, Rmeas and average I/σ(I) were 90.4%, 11.7% and 8.1, respectively, indicating that the data set was sufficient to visualize H and D atoms.

Identification, biochemical characterization and crystallization of the central region of human ATG16L1


ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein–protein interactions essential for autophagosome formation. The ATG12~ATG5–ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12~ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L111–307 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5–ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5–ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72–307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L172–307 subfragment are reported.

Crystallization and X-ray analysis of 23 nm virus-like particles from Norovirus Chiba strain


Norovirus is a major causative pathogen of nonbacterial acute gastroenteritis. Despite the sequence similarity among various strains, noroviruses of different genotypes show different antigenicities and different binding profiles to histo-blood group antigens (HBGAs). To reveal the relationships between the structure of the capsid and the diversity in antigenicity and the HBGA-binding profile, virus-like particles (VLPs) of the Chiba strain that belongs to genogroup I, genotype 4 were crystallized for X-ray structural analysis. Diffraction data were collected and processed at 3.2 Å resolution. The crystal belonged to space group I222, with unit-cell parameters a = 290.0, b = 310.4 c = 350.4 Å. The possible packing model indicated that the diameter of the particle was 280 Å, which was much smaller than the 38 nm VLPs of Norovirus Norwalk strain (NV) with T = 3 icosahedral symmetry and composed of 180 VP1 proteins. The structure was solved by molecular replacement using the structure of the VP1 pentamer of NV 38 nm VLPs as a search model, revealing that the VLPs were smaller particles: 23 nm VLPs with T = 1 icosahedral symmetry, the structure of which has not yet been analyzed at high resolution. The structure of 23 nm VLPs will enable the two different VLPs of Norovirus to be compared, which will provide important information for understanding the structural basis of capsid formation.

Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction


A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Structure, activity and thermostability investigations of OXA-163, OXA-181 and OXA-245 using biochemical analysis, crystal structures and differential scanning calorimetry analysis


The first crystal structures of the class D β-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20 Å resolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07 Å resolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8°C), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4°C) and OXA-181 (Tm = 52.6°C). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.