Publishing structural genomics results continued: the SSGCID Special Issue

Structure of New Delhi metallo-β-lactamase 1 (NDM-1)

Metallo-β-lactamase enzymes confer resistance to a wide variety of commonly used antibiotics; this resistance poses a significant threat to human health where it arises in a pathogenic bacterium. Here, the structure of the novel metallo-β-lactamase NDM-1, which is capable of providing resistance against a broad spectrum of antibacterial agents, has been solved.

Antigen recognition by antibody C836 through adjustment of V_L/V_H packing

Comparison of the Fab structure in the free and antigen-bound form indicates an induced-fit mechanism of IL-13 recognition by antibody C836 through rigid-body rotation of the V_L and V_H domains.

ADP–Mg²⁺ bound to the ATP-grasp domain of ATP-citrate lyase

ATP-citrate lyase catalyzes the production of acetyl-CoA from citrate, CoA and ATP. Crystals were grown of the amino-terminal portion of the human enzyme in the presence of tartrate, ATP and magnesium ions. The crystal structure shows the inhibitor tartrate and the product ADP–Mg²⁺ bound to the protein.

Structures of native and Fe-substituted SOD2 from Saccharomyces cerevisiae

The crystal structures of both native manganese SOD2 and iron-substituted SOD2 from S. cerevisiae were solved. The surface-potential properties showed a similar pattern to those of Cu/Zn-SODs.

Structures of respiratory syncytial virus nucleocapsid protein from two crystal forms: details of potential packing interactions in the native helical form

Structures of the nucleocapsid protein of human respiratory syncytial virus have been obtained from two crystal forms. The packing in one crystal form suggests that ionic interactions contribute to the stability of the native helical form of HRSVN.

Structure of PA4019, a putative aromatic acid de­carboxylase from Pseudomonas aeruginosa

The crystal structure of recombinant UbiX has been determined to 1.5 Å resolution.

Substrate binding of a GH5 endoglucanase from the ruminal fungus Piromyces rhizinflata

The structure of a complex of an endoglucanase with cellotriose was determined with a hexagonal unit cell and showed how the substrate interacts with the enzyme.

Crystallization and preliminary X-ray diffraction studies of sortase A from Streptococcus pneumoniae

Sortase A from S. pneumoniae has been crystallized in two crystal forms: diamond-shaped (construct ΔN₅₉SrtA) and rod-shaped (construct ΔN₈₁SrtA). The diamond-shaped crystals diffracted poorly to 4.0 Å resolution and belonged to a tetragonal system, whereas the rod-shaped crystals diffracted to 2.91 Å resolution and belonged to space group P2₁.

Crystallization and preliminary X-ray crystallographic analysis of the hexameric human p97/VCP ND1 fragment in complex with the UBX domain of human FAF1

The hexameric ND1 fragment of p97/VCP was crystallized in complex with the UBX domain of FAF1. X-ray diffraction data were collected to 3.60 Å resolution and the crystals belonged to space group I222.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120

Alkyl hydroperoxide reductase from Anabaena sp. PCC 7120 was expressed, purified and crystallized. Diffraction data to 2.5 Å resolution revealed that the crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 80, b = 102, c = 109.6 Å.

Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenome­thionine labelling

Crystals of FimP from A. oris were obtained. To facilitate selenomethionine labelling, three methionines were introduced by site-directed mutagenesis.

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

The complex of MoPrP(120–232) and Fab POM1 has been crystallized (space group C2, unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°). Diffraction data to 2.30 Å resolution have been collected using synchrotron radiation.

Crystallization and preliminary X-ray analysis of the Entamoeba histolytica α-actinin-2 rod domain

The rod domain of α-actinin-2 from E. histolytica, the aetiological agent of human amoebiasis, was crystallized and synchrotron diffraction data were collected from crystals vitrified in liquid nitrogen.

Influence of the N-terminal peptide on the cocrystallization of a thermophilic endo-β-1,4-glucanase with polysaccharide substrates

In order to confirm the effect on cocrystallization, two N-terminally truncated variants of a thermostable endoglucanase from the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 were constructed, purified and cocrystallized at 291 K.

Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde de­hydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.)

Crystals of betaine aldehyde dehydrogenase 2 from rice (O. sativa L.) belonged to a C-centred orthorhombic space group and diffraceted X-rays to 2.6 Å resolution.

Cloning, purification and preliminary crystallographic studies of the 2AB protein from hepatitis A virus

The 2AB protein derived from the nonstructural P2 region of hepatitis A virus has been cloned, purified and crystallized. The preliminary characterization of native and selenomethionine-derivative crystals is reported.

Crystallization and preliminary crystallographic studies of Campylobacter jejuni ChuZ, a member of a novel haem oxygenase family

ChuZ–haemin complex crystals belonged to space group C222₁, with unit-cell parameters a = 106.474, b = 106.698, c = 52.464 Å, α = β = γ = 90°. Diffraction data have been collected to 2.4 Å resolution.

Crystallization of a paraspeckle protein PSPC1–NONO heterodimer

A truncated heterodimer of human PSPC1–NONO protein, a paraspeckle-specific complex, has been crystallized and the diffraction data collected to a resolution of 1.9 Å.

Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation

The DHH-family phosphoesterase NrnA is involved in the degradation of small RNA oligomers. B. subtilis NrnA crystallized in space group P2₁ and diffracted X-rays to a resolution of 2.0 Å.

Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily

Yeast Sfh3, a Sec14-like phosphatidylinositol-transfer protein with unique functional properties, has been crystallized. X-ray data sets were collected from native and selenomethionine-substituted crystals to 2.2 and 1.93 Å resolution, respectively.

Crystallization and preliminary crystallographic studies of the HA3 subcomponent of the type B botulinum neurotoxin complex

The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values.

Purification, crystallization and preliminary X-ray crystallographic analysis of PBP4 from Listeria monocytogenes

The crystallization of PBP4 from L. monocytogenes is reported.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

The C-terminal cytosolic domain of mouse anoctamin 1 (mANO1, also known as TMEM16A) was cloned, overexpressed, purified and crystallized. The crystals belonged to space group P2₁2₁2₁ and diffracted to 2.3 Å resolution.

Purification, crystallization and preliminary crystallographic studies of the TLDc domain of oxidation resistance protein 2 from zebrafish

The crystallization of the TLDc domain of oxidation resistance protein 2 from zebrafish is reported.

Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

The successful preparation of a mutant KH domain representing the first KH domain of PCBP1 and its crystallization in complex with a C-rich DNA are reported. This structure is anticipated to provide high-resolution information that will allow better understanding of the basis of cytosine specificity by PCBPs.

Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA

To further understand the structural basis of MmeI (a type IIL restriction enzyme) DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate.

Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties

eCGP123, an extremely stable GFP with photoswitching properties, has been expressed, purified and crystallized. A diffraction data set has been collected at 2.10 Å resolution.

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ⁴-(5α)-de­hydrogenase from Rhodococcus jostii RHA1

The gene for 3-ketosteroid Δ⁴-(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222₁ and diffraction data were collected to a resolution of 1.6 Å.

Cloning, expression, purification, crystallization and preliminary X-ray studies of the C-terminal domain of Rv3262 (FbiB) from Mycobacterium tuberculosis

The C-terminal domain of FbiB, a bifunctional protein that is essential for the biosynthesis of cofactor F₄₂₀ in M. tuberculosis, has been expressed, purified and crystallized. The crystals diffracted to 2.0 Å resolution and were suitable for structure determination.

Crystallization and preliminary X-ray diffraction studies of the transcriptional repressor PaaX, the main regulator of the phenylacetic acid degradation pathway in Escherichia coli W

PaaX, the main regulator of the phenylacetic acid degradation pathway in bacteria, has been crystallized using the sitting-drop vapour-diffusion method. Preliminary X-ray data to 2.3 Å resolution allowed determination of its unit-cell parameters and space group and initiation of its structure determination.

Overexpression, crystallization and preliminary X-ray diffraction analysis of L-ribose isomerase from Acinetobacter sp. strain DL-28

Recombinant L-ribose isomerase from Acinetobacter sp. was crystallized. Diffraction data were collected to 2.2 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of phosphoglycerate kinase from Streptococcus pneumoniae

Phosphoglycerate kinase, a two-domain enzyme involved in the glycolytic pathway, has been crystallized. Both the N-terminal domain and a ternary complex of the full-length enzyme with substrates were crystallized by the sitting-drop vapour-diffusion method. Inclusion of the substrates was critical in order to obtain crystals of the full-length protein.

Crystallization of a challenging antigen–antibody complex: TLR3 ECD with three noncompeting Fabs

Methods that led to the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 ECD with three Fab fragments combined with microseed matrix screening and additive screening.

Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis

Methods for the expression, purification and crystallization of the class C acid phosphatase from M. bovis are reported. This enzyme is atypical in that it is nearly 20 kDa larger than other known class C acid phosphatases.

Humidity control can compensate for the damage induced in protein crystals by alien solutions

Protein crystals typically deteriorate upon soaking in solutions very different from those used to grow them (the `mother liquor'). This is particularly true of solutions containing lower salt concentrations and organic solvents (i.e. DMSO, ethanol). Optimization of the relative humidity environment of the crystals can compensate for these effects and allow recovery of diffraction quality.