Subscribe: Acta Crystallographica Section F
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: 2017-09-01


Crystal structure of the DNA sequence d(CGTGAATTCACG)2 with DAPI


The structure of 4′,6-diamidine-2-phenylindole (DAPI) bound to the synthetic B-DNA oligonucleotide d(CGTGAATTCACG) has been solved in space group P212121 by single-crystal X-ray diffraction at a resolution of 2.2 Å. The structure is nearly isomorphous to that of the previously reported crystal structure of the oligonucleotide d(CGTGAATTCACG) alone. The adjustments in crystal packing between the native DNA molecule and the DNA–DAPI complex are described. DAPI lies in the narrow minor groove near the centre of the B-DNA fragment, positioned over the A–T base pairs. It is bound to the DNA by hydrogen-bonding and van der Waals interactions. Comparison of the two structures (with and without ligand) shows that DAPI inserts into the minor groove, displacing the ordered spine waters. Indeed, as DAPI is hydrophobic it confers this behaviour on the DNA and thus restricts the presence of water molecules.

HicAB toxin–antitoxin complex from Escherichia coli: expression and crystallization


Toxin–antitoxin (TA) systems are widespread in both bacteria and archaea, where they enable cells to adapt to environmental cues. TA systems play crucial roles in various cellular processes, such as programmed cell death, cell growth, persistence and virulence. Here, two distinct forms of the type II toxin–antitoxin complex HicAB were identified and characterized in Escherichia coli K-12, and both were successfully overexpressed and purified. The two proposed forms, HicABL and HicABS, differed in the presence or absence of a seven-amino-acid segment at the N-terminus in the antitoxin HicB. The short form HicABS readily crystallized under the conditions 0.1 M Tris–HCl pH 8.0, 20%(w/v) PEG 6000, 0.2 M ammonium sulfate. The HicABS crystal diffracted and data were collected to 2.5 Å resolution. The crystal belonged to space group I222 or I212121, with unit-cell parameters a = 67.04, b = 66.31, c = 120.78 Å. Matthews coefficient calculation suggested the presence of two molecules each of HicA and HicBS in the asymmetric unit, with a solvent content of 55.28% and a Matthews coefficient (VM) of 2.75 Å3 Da−1.

3,6-Anhydro-l-galactonate cycloisomerase from Vibrio sp. strain EJY3: crystallization and X-ray crystallographic analysis


3,6-Anhydro-l-galactonate cycloisomerase (ACI), which is found in the marine bacterium Vibrio sp. strain EJY3, converts 3,6-anhydro-l-galactonate into 2-keto-3-deoxygalactonate. ACI is a key enzyme in the metabolic pathway of 3,6-anhydro-l-galactose (AHG). Study of AHG metabolism is important for the efficient fermentation of agar and biofuel production, because AHG is a sugar that is non-fermentable by commercial microorganisms. The aci gene from Vibrio sp. strain EJY3 was cloned, and the recombinant protein was overexpressed and crystallized in order to determine the structure and understand the function of the protein. The crystals diffracted to 2.2 Å resolution and belonged to space group P41212 or P43212, with unit-cell parameters a = b = 87.9, c = 143.5 Å. The Matthews coefficient was 2.3 Å3 Da−1, with a solvent content of 47%.

Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana


Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, from Cladophialophora batiana was expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35°C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determined to 1.75 Å resolution. The active-site compositions of CbZHD and ZHD101 were analyzed.

The LRR-Roc-COR module of the Chlorobium tepidum Roco protein: crystallization and X-ray crystallographic analysis


Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacterium Chlorobium tepidum has been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of the C. tepidum Roco protein is reported. The LRR-Roc-COR crystals belonged to space group P212121, with unit-cell parameters a = 95.6, b = 129.8, c = 179.5 Å, α = β = γ = 90°, and diffracted to a resolution of 3.3 Å. Based on the calculated Matthews coefficient, Patterson map analysis and an initial molecular-replacement analysis, one protein dimer is present in the asymmetric unit. The crystal structure of this protein will provide valuable insights into the interaction between the Roc-COR and LRR domains within Roco proteins.

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO


The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory.

Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis


Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Å resolution and were indexed in space group P1, with unit-cell parameters a = 41.9, b = 48.1, c = 62.2 Å, α = 97.3, β = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å3 Da−1 and a solvent content of 50.97%.

Production, crystallization and structure determination of a mycobacterial glucosylglycerate hydrolase


Glucosylglycerate hydrolase is highly conserved among rapidly growing mycobacteria and has been found to be involved in recovery from nitrogen starvation by promoting the rapid mobilization of the glucosylglycerate that accumulates under these conditions. Here, the production, crystallization and structure determination of glucosylglycerate hydrolase from Mycobacterium hassiacum using two-wavelength anomalous diffraction of selenomethionine-substituted crystals are described. The monoclinic (space group P21) crystals diffracted to ∼2.0 Å resolution at a synchrotron-radiation source and contained four molecules in the asymmetric unit, corresponding to a Matthews coefficient of 3.07 Å3 Da−1 and a solvent content of 59.9%. The quality of the experimental phases allowed the automated building of 1677 of the 1792 residues in the asymmetric unit.