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Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges.

A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V M = 2.5 Å3 Da−1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.

A β-glucosidase A (BglA) from the thermophile Halothermothrix orenii has been cloned, purified and crystallized in an orthorhombic space group. X-ray diffraction data have been collected to 3.5 Å resolution, and the structure was solved by molecular replacement, revealing the presence of two molecules in the asymmetric unit.

The β-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.

ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution.

The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12 Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V M value of 2.46 Å3 Da−1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V M of 4.80 Å3 Da−1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.

Crystals of the Grb2 SH2 domain in complex with a phosphotyrosyl peptide corresponding to residues 921–930 of focal adhesion kinase (FAK) have been obtained using the sitting-drop vapour-diffusion technique. Data have been collected to 2.49 Å resolution.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2–FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921–930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3121, with unit-cell parameters a = b = 102.7, c = 127.6 Å, α = β = 90.0, γ = 120.0°. A diffraction data set was collected from a flash-cooled crystal at 100 K to 2.49 Å resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress.

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase from A. mylitta are reported.

Glyceraldehyde-3-phosphate dehydrogenase from Antheraea mylitta (AmGAPDH) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 85.81, b = 133.72, c = 220.37 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.2 Å. The presence of three molecules in the asymmetric unit gave a Matthews coefficient (V M) of 2.80 Å3 Da−1, with a solvent content of 56.08%.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a “right-turn” elongated bundle; the globular fold is mainly maintained by hydrophobic interactions. The bound peptide further stabilizes the structure. Certain signaling events such as phosphorylation and molecule interplay may induce opening of the helix bundle. Such conformational change is proposed to precede departure of FAK from focal adhesions, which starts focal adhesion turnover.

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with Grb2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH dependent, but this does not reflect the pH dependence of Src kinase activity. CD and NMR data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the μs-ms time scale.

Orthorhombic crystals of mouse 3(17)α-hydroxysteroid dehydrogenase were obtained from buffered polyethylene glycol solutions. The crystals diffracted to a resolution of 1.8 Å at the Swiss Light Source beamline X06SA.

The 3(17)α-hydroxysteroid dehydrogenase from mouse is involved in the metabolism of oestrogens, androgens, neurosteroids and xenobiotic compounds. The enzyme was crystallized by the hanging-drop vapour-diffusion method in space group P2221, with unit-cell parameters a = 84.91, b = 84.90, c = 95.83 Å. The Matthews coefficient (V M) and the solvent content were 2.21 Å3 Da−1 and 44.6%, respectively, assuming the presence of two molecules in the asymmetric unit. Diffraction data were collected to a resolution of 1.8 Å at the Swiss Light Source beamline X06SA using a MAR CCD area detector and gave a data set with an overall R merge of 6.8% and a completeness of 91.1%.

Crystals of the human Plk1 Polo-box domain in complex with a Cdc25C target peptide in an unphosphorylated and a phosphorylated state have been obtained in orthorhombic and monoclinic forms that diffract to 2.1 and 2.85 Å, respectively, using synchrotron radiation.

Polo-like kinase (Plk1) is crucial for cell-cycle progression via mitosis. Members of the Polo-like kinase family are characterized by the presence of a C-terminal domain termed the Polo-box domain (PBD) in addition to the N-terminal kinase domain. The PBD of Plk1 was cloned and overexpressed in Escherichia coli. Crystallization experiments of the protein in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C yield crystals suitable for X-­ray diffraction analysis. Crystals of the PBD in complex with the phosphorylated peptide belong to the orthorhombic space group P212121, with unit-cell parameters a = 38.23, b = 67.35, c = 88.25 Å, α = γ = β = 90°, and contain one molecule per asymmetric unit. Crystals of the PBD in complex with the unphosphorylated peptide belong to the monoclinic space group P21, with unit-cell parameters a = 40.18, b = 49.17, c = 56.23 Å, α = γ = 90, β = 109.48°, and contain one molecule per asymmetric unit. The crystals diffracted to resolution limits of 2.1 and 2.85 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS), respectively.

l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis.

l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P212121, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å3 Da−1. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

Single orthorhombic crystals of M. genitalium protein MG289 have been grown and shown to diffract X-rays to 2.8 Å resolution with good statistics. The structure obtained from these data will help to provide insight into the function of the protein as well as improving the understanding of its role in this human pathogen.

Mycoplasma genitalium is a human pathogen that is associated with non­gonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 Å resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 Å. The diffraction data after processing had an overall R merge of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P212121 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å.

Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive ortho­rhombic space group P212121, with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M) of 2.85 Å3 Da−1 and a solvent content of 57%.

The putative thiamine-biosynthesis protein PH1313 from P. horikoshii OT3 was overexpressed, purified and crystallized. The crystals belong to space group P212121 and diffract X-rays to 1.9 Å resolution.

The putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 has been overexpressed and purified. Crystallization was performed by the oil-microbatch method using 28%(v/v) 2-methyl-2,4-pentanediol as a precipitant at 291 K. A native X-ray diffraction data set at 1.9 Å resolution and a single anomalous dispersion data set from a selenomethionine-derivative crystal at 2.1 Å resolution were collected using synchrotron radiation at 100 K. The native crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 71.7, b = 71.2, c = 141.8 Å.

Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.

The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R merge of 0.12. Further experimental phasing methods are under way.

The molybdate-binding protein (ModA) from X. axonopodis pv. citri was crystallized with sodium molybdate in the presence of PEG or sulfate. The crystal diffracted to a maximum resolution of 1.7 Å and belongs to the orthorhombic space group C2221, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å.

Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 Å using synchrotron radiation. The crystal belongs to the orthorhombic space group C2221, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.

Crystals of arylmalonate decarboxylase from A. bronchisepticus were obtained which diffracted X-rays to a resolution of at least 3.0 Å.

Arylmalonate decarboxylase catalyses the enantioselective decarboxylation of α-aryl-α-methylmalonates to produce optically pure α-arylpropionates. The enzyme was crystallized with ammonium sulfate under alkaline pH conditions with the aim of understanding the mechanism of the enantioselective reaction. X-ray diffraction data collected to a resolution of 3.0 Å at cryogenic temperature showed that the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 83.13, b = 99.62, c = 139.64 Å. This suggested that the asymmetric unit would contain between four and six molecules. Small-angle X-ray scattering revealed that the enzyme exists as a monomer in solution. Thus, the assembly of molecules in the asymmetric unit was likely to have been induced during the crystallization process.

The adhesive domain of SdrE from Staphylococcus aureus was recombinantly expressed in Escherichia coli and crystallized. X-ray diffraction data were collected to 1.8 Å resolution.

The adhesive domain of SdrE from Staphylococcus aureus was recombinantly expressed in Escherichia coli. The purified protein was identified by SDS–PAGE and MALDI–TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 8000 as the primary precipitating agent. X-ray diffraction data were collected to 1.8 Å resolution from a single crystal of the protein. Preliminary X-ray analysis indicated that the crystal belonged to space group P1, with unit-cell parameters a = 40.714, b = 66.355, c = 80.827 Å, α = 111.19, β = 93.99, γ = 104.39°.

The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal.

The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V M of 1.8 Å3 Da−1.

Mouse peroxiredoxin II was crystallized in an orthorhombic space group and native X-ray diffraction data were collected.

Peroxiredoxin II was cloned from mouse B cells into pCold 1 expression vector and produced as a His-tagged recombinant protein in Escherichia coli. A ring form was isolated by gel filtration. A crystal obtained by the sitting-drop vapour-diffusion method diffracted to 1.77 Å resolution at 100 K. The crystal belonged to space group P21212, with unit-cell parameters a = 117.4, b = 133.9, c = 139.1 Å. The asymmetric unit is expected to contain six dimers of peroxiredoxin II, with a corresponding solvent content of 39.3%. Peaks in the native Patterson function together with pseudo-systematic absences suggested that the crystals suffered from severe translational pseudosymmetry.

A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The space group of the crystals was determined as primitive orthorhombic P212121, with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 Å.

A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The crystals diffracted X-rays to beyond 2.0 Å resolution using a synchrotron-radiation source. The space group of the crystals was determined as primitive orthorhombic P212121, with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 Å. The crystals contain one molecule in the asymmetric unit (V M = 2.5 Å3 Da−1) and have a solvent content of 50%.

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

A highly diversified novel immune-type receptor from catfish, NITR10, was crystallized to reveal novel mechanisms of immune recognition.

X-ray diffraction data from crystals of a novel immune-type receptor (NITR10 from the catfish Ictalurus punctatus) were collected to 1.65 Å resolution and reduced to the primitive hexagonal lattice. Native and selenomethionine derivatives of NITR10 crystallized under different conditions yielded P3121 crystals. SeMet NITR10 was phased to a correlation coefficient of 0.77 by SAD methods and experimental electron-density maps were calculated to 1.65 Å. Five NITR10 molecules are predicted to be present in the asymmetric unit based on the Matthews coefficient.

N. tabacum class V chitinase has been crystallized. X-ray diffraction data were collected to 1.2 Å resolution using a synchrotron-radiation source.

The plant chitinases, which have been implicated in self-defence against pathogens, are divided into at least five classes (classes I, II, III, IV and V). Although the crystal structures of several plant chitinases have been solved, no crystal structure of a class V chitinase has been reported to date. Here, the crystallization of Nicotiana tabacum class V chitinase (NtChiV) using the vapour-diffusion method is reported. The NtChiV crystals diffracted to 1.2 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 62.4, b = 120.3, c = 51.9 Å. The asymmetric unit of the crystals is expected to contain one molecule.